Methods and Compositions for Modulating TH-GM Cell Function

ABSTRACT

Disclosed herein is a T-helper cell (“T H -GM” cell) that is regulated by IL-7/STAT5 and which secrete GM-CSF/IL-3. Also disclosed are methods and compositions for modulating T H -GM function for the treatment of, e.g., inflammatory disorders. Diagnostic and prognostic methods for specifically identifying T H -GM-mediated inflammatory disorders (e.g., rheumatoid arthritis), as distinct from and/or in addition to non-T H -GM-mediated (e.g., TNF-α-mediated) inflammatory disorders, are also provided.

RELATED APPLICATION

This application claims the benefit of Singapore Patent Application No.10201406130P, filed Sep. 26, 2014. The entire teachings of the aboveapplication are incorporated herein by reference.

BACKGROUND OF THE INVENTION

A significant body of research has led to the current model of immunityand inflammation, as well as the dysregulation in immune andinflammatory disorders. It is currently understood that CD4⁺ helper T(T_(H)) cells play a crucial role in host defense against variouspathogens by orchestrating adaptive and innate immune responses. UponT-cell receptor (TCR) activation by cognate antigen, naïve CD4⁺ T cellsare committed to differentiate into at least live major subsets: T_(H)1,T_(H)2, T_(H)17, iT_(reg) and T_(FH), which are modulated by cytokinemilieus. T_(H)1 and T_(H)17 cells are known to be the primary effectorsof inflammation. However, the pathogenic roles of either T_(H)1 orT_(H)17 in various inflammatory disorders remain unclear. For example,recent studies conflict with previously understood paradigm of T_(H)17in multiple sclerosis (MS) pathogenicity (Haak et al., 2009), making itmore challenging to identify potential drug targets for MS therapy.Similarly, while rheumatoid arthritis (RA) is traditionally understoodto be a disorder mediated by tumor necrosis factor α (TNF-α), up to 40%of RA patients fail to respond to anti-TNF-α treatment.

Accordingly, there remains a significant unmet need for effectivetreatment methods for autoimmune and inflammatory disorders such as,e.g., MS and RA.

SUMMARY OF THE INVENTION

The present disclosure relates, in part, to the identification of aninterleukin-7 (IL-7)/signal transducer and activator of transcription 5(STAT5)-regulated granulocyte macrophage colony-stimulating factor(GM-CSF)/IL-3-producing T_(H) cells, termed T_(H)-GM, which represent adistinct helper T cell subset with unique developmental and functionalcharacteristics. Identified herein is an inflammatory pathway mediatedby T_(H)-GM cells (T_(H)-GM-mediated inflammatory pathway), whichrepresents an independent inflammatory pathway apart from knownnon-T_(H)-GM-mediated inflammatory pathways (e.g., TNF-a, IL-6, andIL-1b pathways of inflammation). The present disclosure provides methodsand compositions for diagnosing inflammatory disorders that areT_(H)-GM-mediated, and modulating T_(H)-GM cell function for thetreatment of inflammatory disorders mediated by the T_(H)-GM pathway.

Accordingly, in one aspect, the present disclosure provides a method ofdiagnosing a T_(H)-GM-mediated inflammatory disorder in a patientsuffering from an inflammatory disorder, comprising: a) contacting asample collected from a patient suffering from an inflammatory disorderwith a detecting agent that detects a polypeptide or nucleic acid levelof STAT5 (e.g., phospho-STAT5 (Tyr694)). IL-7, GM-CSF or IL-3, or acombination thereof; and h) quantifying the polypeptide or nucleic acidlevel of STAT5 (e.g., phospho-STAT5 (Tyr694)), IL-7, GM-CSF or IL-3, ora combination thereof, wherein an increased level of STAT5 (e.g.,phospho-STAT5 (Tyr694)), interleukin-7 (IL-7), GM-CSF or interleukin-3(IL-3), or a combination thereof relative to a reference level indicatesthat the patient suffers from a T_(H)-GM-mediated inflammatory disorder.

In another aspect, the present disclosure provides an isolatedpopulation of GM-CSF-secreting T-helper cells (T_(H)-GM), wherein theT_(H)-GM cells are differentiated from cluster of differentiation 4(CD4+) precursor cells in the presence of IL-7 and activated STAT5, andwherein the T_(H)-GM cells express GM-CSF and IL-3.

In another aspect, the present disclosure provides a method ofmodulating T_(H)-GM function, comprising contacting the T_(H)-GM, orCD4+ precursor cells, or both, with a modulating agent that modulatesT_(H)-GM function.

In some aspects, the present disclosure provides a method of treating aT_(H)-GM-mediated inflammatory disorder in a patient in need thereof,comprising administering to said patient an effective amount of amodulating agent that modulates T_(H)-GM cell function.

In other aspects, the present disclosure provides a method of treatingrheumatoid arthritis in a patient who exhibits limited response toanti-tumor necrosis factor alpha (TNF-α) therapy, comprisingadministering to said patient an effective amount of a modulating agentthat modulates T_(H)-GM function.

In another aspect, the present disclosure provides a method of treatinga STAT5-mediated inflammatory disorder in a patient in need thereof,comprising administering to the patient an effective amount of an agentthat modulates STAT5 function.

In further aspects, the present disclosure provides a method ofscreening to identify a modulator of T_(H)-GM cell function, comprisingcontacting an isolated population of T_(H)-GM cells, or an isolatedpopulation of CD4+ precursor cells, with a candidate agent, anddetermining a readout of T_(H)-GM function in the presence or absence ofthe candidate agent, wherein a change in the readout of T_(H)-GMfunction indicates that the candidate agent is a modulator of T_(H)-GMfunction.

The present disclosure enables the identification or classificationbetween inflammatory disorders that are either primarilyT_(H)-GM-mediated, or primarily non-T_(H)-GM-mediated (e.g., mediated byTNF-α, IL-6, and/or IL-1β), or both. Thus, using the methods describedherein, it is possible to determine whether a patient suffering from,e.g., RA, suffers from an RA that is primarily T_(H)-GM-mediated, orprimarily non-T_(H)-GM-mediated, or both. This differentiation allowsfor a more targeted and tailored method of treating inflammatorydisorders such as RA, for which current treatments are only 40%effective. Further, the present disclosure provides methods andcompositions for prognosing the progression of an inflammatory disorderso as to tailor the treatment according to the stage of the disease.Also provided herein are compositions and methods for and the treatmentof inflammatory disorders, particularly those that areT_(H)-GM-mediated.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing will be apparent from the following more particulardescription of example embodiments of the invention.

FIGS. 1A-1D depict Stat5-conditional mutant mice are resistant to EAE.Clinical EAE scores (FIG. 1A) and incidence (FIG. 1B) of Stat5^(+/+) andStat5^(−/−) mice immunized twice with MOG₃₅₋₅₅/CFA. Data arerepresentative of three independent experiments (FIG. 1A) or pooled fromthree experiments (FIG. 1B, n=18 per group). Clinical scores of EAE miceimmunized once with MOG₃₅₋₅₅/CFA (FIG. 1C, n=5 per group) or immunizedtwice with MOG₃₅₋₅₅/LPS (FIG. 1D). Data are representative of twoindependent experiments.

FIGS. 2A-2D depict reduced neuroinflammation in Stat5 conditional mutantmice. Histology of spinal cord sections obtained from EAE mice at day 9after 2^(nd) immunization (FIG. 1A). Images shown are representative oftwo independent experiments with three mice per group. Scale bars, 200μm (top), 50 μm (bottom). CD4⁺ and CD11b⁺ cells in spinal cord sectionswere stained by immunofluorescence (FIG. 1B). Images shown arerepresentative of two independent experiments with three mice per group.Scale bars, 200 μm. CNS mononuclear cells were analyzed by flowcytometry at peak of disease (FIGS. 2C and 2D). Right panels are cellproportions (FIG. 2C, right) or cell numbers (FIG. 2D, right) pooledfrom two experiments (n=9).

FIGS. 3A and 3B depict the resistance to EAE in Stat5-deficient mice isindependent of T_(H)1, T_(H)17 or T_(reg) cells. Flow cytometricanalysis of IL-17 and IFN-γ expression by CNS-infiltrating CD4⁺ T cellsat peak of disease (FIG. 3A). Data are representative of threeindependent experiments. Percentage of CD25⁺ among CD4⁺ T cells in theCNS of Stat5^(+/+) and Stat5^(−/−) EAE mice at peak of disease wereanalyzed by flow cytometry (FIG. 3B).

FIGS. 4A-4C depict conditional Stat5 mutant mice have no defect in CD4⁺T cell generation in periphery. Spleens were obtained fromMOG₃₅₋₅₅/CFA-immunized Stat5^(+/+) and Stat5^(−/−) mice at day 7 (FIG.4A) and day 21 (FIG. 4B). The proportions of CD4⁺ and CD8⁺ T cells wereanalyzed by flow cytometry. The absolute number of CD4⁺ T cells wascalculated (right panels). Data are representative of two independentexperiments (FIG. 4A) or pooled from two independent experiments (FIG.4B). IL-17 and IFN-γ expression by splenic CD4⁺ T cells of Stat5^(+/+)and Stat5^(−/−) EAE mice was determined by intracellular cytokinestaining (FIG. 4C). Data are representative of three independentexperiments. *p<0.5, **p<0.005, ***p<0.0005.

FIGS. 5A-5D depict Stat5-deficient CD4⁺ T cells can infiltrate CNS butfail to induce effective neuroinflammation. CCR6, CXCR3 and CD69expression by splenic CD4⁺ T cells of Stat5^(+/+) and Stat5^(−/−) EAEmice was measured. Data are representative of two independentexperiments with three to five mice per group (FIG. 5A).CNS-infiltrating CD4⁺ T cells were analyzed at day 7, 9 and 21 afterfirst MOG₃₅₋₅₅/CFA immunization (FIGS. 5B-5D). Cell numbers werecalculated (FIG. 5D). Data are representative of two independentexperiments with three mice per group. *p<0.5.

FIGS. 6A-6C show resistance to EAE in Stat5^(−/−) mice is not caused byany defect in the survival of CD4⁺ T cells in the absence of STAT5. CD4⁺T cell infiltration (FIG. 6A) and clinical scores (FIG. 6B) ofRag2^(−/−) recipient mice transferred with different numbers ofStat5^(+/+) and Stat5^(−/−) CD4⁺ T cells. Clinical scores andfrequencies of CD4⁺ T cells in the CNS at day 21 (disease peak) of EAEinduction (FIG. 6C). *p<0.05. ***p<0.0005.

FIGS. 7A-7C depict the intrinsic defect of Stat5-deficient CD4⁺ T cellsin encephalitogenicity. Clinical EAE scores (FIG. 7A) and incidence(FIG. 7B) of Rag2^(−/−) mice (n=5 per group) after adoptive transfer of2 million MOG₃₅₋₅₅-specific Stat5^(+/+) or Stat5^(−/−) CD4⁺ T cellsrespectively. IL-17 and IFN-γ expression by CNS-infiltrating CD4⁺ Tcells was measured at peak of disease (FIG. 7C). Data represent twoindependent experiments, *p<0.05.

FIGS. 8A-8D depict the diminished induction of GM-CSF in splenicStat5^(−/−) CD4⁺ T cells. In FIGS. 8A-8D, splenocytes were obtained fromMOG₃₅₋₅₅/CFA-immunized Stat5^(+/+) and Stat5^(−/−) mice (n=3 per group)before disease onset and challenged with MOG₃₅₋₅₅ at variousconcentrations for 24 h. GM-CSF secretion was measured by ELISA (FIG.8A). Golgiplug was added in the last 4 h of MOG₃₅₋₅₅ (20 μg/ml)challenge and the frequencies of IL-17⁺ and GM-CSF⁺ cells amongCD4⁺CD44^(hi) T cells were measured (FIG. 8B). In FIGS. 8C and 8C,splenocytes were obtained from MOG₃₅₋₅₅/CFA-immunized Stat5^(−/−),Stat3^(−/−) or wild-type control mice and stimulated with PMA/Ionomycinin the presence of Golgiplug for 4 h. The frequencies of IL-17⁺ andGM-CSF⁺ cells among splenic CD4⁺CD44^(hi) T cells were measured byintracellular cytokine staining. *p<0.05, ***p<0.001.

FIGS. 9A-9C depict the diminished induction of GM-CSF inCNS-infiltrating Stat5^(−/−) CD4⁺ T cells. In FIG. 9A, IL-17, IFN-γ andGM-CSF expression by CNS-infiltrating CD4⁺ T cells of Stat5^(+/+) andStat5^(−/−) mice was measured at peak of disease. The percentage ofGM-CSF⁺ cells among IL-17⁺ or IFN-γ⁺ cells was calculated (bottom right.FIG. 9A). IL-17. IFN-γ and GM-CSF expression by CNS-infiltrating CD4⁺ Tcells of Rag2^(−/−) recipient mice at peak of disease in adoptivetransfer EAE (FIG. 9B). Time-course analysis of cytokine mRNA expressionin the CNS of naïve and MOG₃₅₋₅₅/CFA-immunized Stat5^(+/+) and Stat5−/−mice (n=3 per group at each time point). The RT-PCR data were normalizedto Rn18S, and expression in naïve mice was set to 1 (FIG. 9C). Datarepresent two independent experiments. *p<0.05.

FIGS. 10A-10C show STAT5-mediated GM-CSF induction is independent ofIL-23 or IL-1β signaling. In FIG. 10A, purified CD4⁺ T cells werecultured with TGF-β and IL-6 for 3 days, followed by starvation for 6 h.Then cells were treated with various cytokines for 30 min. and pSTAT3and pSTAT5 was determined by immunoblotting. STAT3 and STAT5 werefurther detected after stripping. FIG. 10B shows the mRNA expression ofIL-23R and IL-1R1 in splenic CD4⁺ T cells of Stat5^(+/+) and Stat5^(−/−)EAE mice (n=3 per group). The RT-PCR data were normalized to β-Actin. InFIG. 10C, splenocytes were obtained from MOG₃₅₋₅₅/CFA-immunized WT micebefore disease onset and challenged with MOG₃₅₋₅₅ (20 μg/ml) in theabsence or presence of IL-2 for 48 h. The frequencies of GM-CSF⁺ andIL-17⁺ cells among CD4⁺CD44^(hi) T cells were measured by flowcytometry. *p<0.05.

FIGS. 11A-11C depict IL-7-induced STAT5 activation promotes GM-CSFexpression in autoreactive CD4⁺ T cells. Splenocytes were obtained fromMOG₃₅₋₅₅/CFA-immunized Stat5^(+/+) and Stat5^(−/−) mice before diseaseonset and challenged with MOG₃₅₋₅₅ (20 μg/ml) in the absence or presenceof IL-7 for 48 h. Frequencies of GM-CSF⁺ and IL-17⁺ cells amongCD4⁺CD44^(hi) T cells were measured by flow cytometry (FIG. 11A). GM-CSFsecretion was measured by ELISA (FIG. 11B). Data represent twoindependent experiments with two to three mice per group. SplenicCD62L^(hi)CD44^(lo) and CD62L^(lo)CD44^(hi) T cells fromMOG₃₅₋₅₅/CFA-immunized mice were sorted out. Cells were stimulated withanti-CD3 and anti-CD28 in the absence or presence of IL-7 for 4 h andthen harvested for the analysis of GM-CSF expression by RT-PCR (FIG.11C). *p<0.05

FIGS. 12A-12F depict IL-7Rα neutralization attenuates GM-CSF expressionand ameliorates EAE. Clinical scores of EAE mice (n=5) treated withanti-IL-7Rα or normal IgG given every other day from day 5 after 2^(nd)immunization, as indicated by arrows. Data represent two independentexperiments (FIG. 12A). Spinal cord sections were obtained from EAE miceat day 11 after 2^(nd) immunization. Immune cell infiltration wasassessed histologically. Images shown are representative of threeindividuals per group. Scale bars, 200 μm (top), 50 μm (FIG. 12B,bottom). The percentages of CD4⁺ and CD8⁺ T cells in spleens of EAEmice. Data represent two independent experiments (FIG. 12C). FIGS. 12Dand 12E illustrate the frequencies of GM-CSF⁺, IL-17⁺ and IFN-γ⁺ cellsamong CD4⁺ T cells in the CNS of EAE mice receiving different treatment.The mRNA expression of IFN-γ, IL-17 and GM-CSF in the CNS of EAE mice(FIG. 12F). *p<0.05

FIGS. 13A and 13B depict the differentiation of GM-CSF-expressing T_(H)cells is distinct from T_(H)17 and T_(H)1. Naïve CD4⁺ T cells wereprimed with plate-bound anti-CD3 and soluble anti-CD28 in the presenceof a combination of various cytokines and neutralizing antibodies asindicated. GM-CSF, IL-17 and IFN-γ expression was analyzed byintracellular staining (FIG. 13A) or RT-PCR (FIG. 13B)

FIGS. 14A-14D show the effect of IL-2 and IL-6 on T_(H)-GMdifferentiation from naïve T cells. GM-CSF and IFN-γ expression in naiveCD4⁺ T cells activated for 72 h with anti-CD3 alone or plus anti-CD28(FIG. 14A). In FIG. 14B, sorted naïve CD4⁺ T cells were stimulated withanti-CD3 and anti-CD28 in the presence of neutralizing antibodiesagainst IL-12 and IFN-γ without or with the addition of IL-6. Thefrequencies of GM-CSF⁺ and IL-17⁺ cells were measured by intracellularstaining (FIG. 14B). In FIG. 14C, naïve CD4⁺ T cells from Stat3^(+/+)and Stat3^(−/−) mice were polarized under conditions as indicated for 72h. The frequencies of GM-CSF⁺ and IL-17⁺ cells were analyzed. In FIG.14D, naïve CD4⁺ T cells were activated with anti-CD3 and anti-CD28 inthe presence of IL-2 or anti-IL-2. The frequencies of GM-CSF⁺, IL-17⁺and IFN-γ⁺ cells were analyzed.

FIGS. 15A-15F depict IL-7-STAT5 signaling programs T_(H)-GMdifferentiation from naïve precursor cells. Naïve CD4⁺ T cells wereprimed with plate-hound anti-CD3 and soluble anti-CD28 in the presenceof various concentration of IL-7 as indicated. GM-CSF and IFN-γexpression was analyzed by intracellular staining (FIG. 15A) or ELISA(FIG. 15B). In FIGS. 15C and 15D, Stat5^(+/+) and Stat5^(−/−) naïve CD4⁺T cells were activated with anti-CD3 and anti-CD28 in the presence IL-7for 3 days. GM-CSF, IL-17 and IFN-γ expression was analyzed byintracellular cytokine staining (FIG. 15C). GM-CSF secretion wasmeasured by ELISA (FIG. 15D). Immunoblotting of pSTAT5 and STAT5 inIL-7-stimulated CD4⁺ T cells isolated from Stat5^(−/−) or control mice(FIG. 15E). The ChIP assay was performed with Stat5^(+/+) andStat5^(−/−) CD4⁺ T cells using normal IgG or STAT5-specific antibody.The binding of antibodies to Csf2 promoter region was detected by RT-PCR(FIG. 15F).

FIGS. 16A and 16B depict the differentiation conditions for T_(H)-GMsubset. Naïve CD4⁺ T cells were activated with anti-CD3 and anti-CD28 inthe presence of IL-7 or/and anti-IFN-γ as indicated. GM-CSF. IL-17 andIFN-γ expression was analyzed (FIG. 16A). The mRNA expression of T-betand RORγt in naïve. T_(H)1 (IL-12+anti-IL-γ), T_(H)17(TGF-β+IL-6+anti-IFN-γ+anti-IL-4) and T_(H)-GM cells (IL-7 anti-IFN-γ)(FIG. 16B). The RT-PCR data were normalized to Gapdh, and expression innaïve T cells was set to 1.

FIGS. 17A-17E illustrate that IL-7 but not IL-2 induces STAT5 activationand GM-CSF expression in naïve CD4⁺ T cells. FIGS. 17A-17C show flowcytometry of CD25 and CD127 on the surface of naïve CD4⁺ T cells orcells activated with anti-CD3 and anti-CD28 at various time points asindicated. Activation of STAT5 in naïve CD4⁺ T cells stimulated withIL-2 or IL-7 for 30 min (FIG. 17D). FIG. 17E shows the mRNA expressionof GM-CSF in naïve CD4⁺ T cells stimulated with anti-CD3 and anti-CD28in the presence of IL-2 or IL-7. The RT-PCR data were normalized toβ-Actin, and expression in naïve T cells activated for 2 h withoutcytokine was set to 1.

FIGS. 18A-18C show that both IL-2 and IL-7 can induce STAT5 activationand GM-CSF expression in activated CD4⁺ T cells. As shown in FIGS. 1.8Aand 18B, CD4⁺ T cells were activated with anti-CD3 and anti-CD28 for 3days. After resting in fresh medium, cells were stimulated with IL-2 orIL-7 at various time points. The pTyr-STAT5 and β-Actin were detected byimmunoblotting (FIG. 18A). GM-CSF mRNA expression was measured by RT-PCR(FIG. 18B). The RT-PCR data were normalized to 13-Actin, and expressionin cells without cytokine stimulation was set to 1. The ChIP assay shownin FIG. 18C was performed with normal IgG or STAT5-specific antibody.The binding of antibodies to Csf2 promoter region was detected byRT-PCR.

FIG. 19 depicts surface molecules selectively expressed at high level orlow level in T_(H)-GM subset as characterized by microarray analysis.These surface molecules specific for each lineage serves as markers,signatures and potential targets for novel diagnosis, treatment andprevention of autoimmune inflammation including, but not limited tomultiple sclerosis and rheumatoid arthritis. These cell surfacemolecules are listed in detail in Table 1. The order of naïve, Th1,Th17, and Th-GM as indicated in the figure insert is the same as thatappears for the bars in each graph.

FIGS. 20A-20D show that IL-3 is preferentially expressed in T_(H)-GMcells. In FIGS. 20A and 20B, naïve CD4⁺ T cells were activated withanti-CD3 and anti-CD28 under T_(H)1—(IL-12+anti-IL-4),T_(H)17−(TGF-β+IL-6+anti-IFN-γ+anti-IL-4) and T_(H)-GM—(GM-CSF⁺ T_(H),IL-7+anti-IFN-γ+anti-IL-4) polarizing conditions. GM-CSF and IL-3expression was analyzed by intracellular staining (FIG. 20A). The mRNAexpression of IL-3, EBI-3, PENK or RANKL cytokines was measured byRT-PCR (FIG. 20B). Frequency of IL-3+ cells differentiated without orwith IL-7 (FIG. 20C). GM-CSF and IL-3 expression by WT orSTAT5-deficient GM-CSF-producing TH cells (FIG. 20D).

FIG. 21 depicts clinical EAE scores of Rag2^(−/−) mice (n=3˜6 mice pergroup) after adoptive transfer of 6×10⁵ various MOG₃₅₋₅₅-specific T_(H)subsets.

FIGS. 22A-27E depict inhibition of STAT5 activation suppresses T_(H)-GMcell differentiation in vitro. CD4⁺ T cells were pre-incubated withSTAT5 inhibitor (Calbiochem) (FIG. 22A) or JAK3 inhibitor (FIG. 22B) atindicated concentrations or vehicle (−) for 1 h before stimulation withIL-7 for 30 min. Activation (Tyr694 phosphorylation) of STAT5 wasdetermined by immunoblotting. CD4⁺ T cells were pre-incubated with STAT5inhibitor at indicated concentrations or vehicle (−) for 1 h beforestimulation with IL-6 for 30 min. Activation (Tyr705 phosphorylation) ofSTAT3 was determined by immunoblotting (FIG. 22C). In FIG. 221), CD4⁺ Tcells were pre-incubated with STAT5 inhibitor at indicatedconcentrations or vehicle (−) for 1 h before stimulation with IFN-γ for30 min. Activation (Tyr701 phosphorylation) of STAT1 was determined byimmunoblotting. In FIG. 22E, naïve CD4⁺ T cells were isolated andactivated under neutral condition or T_(H)-GM cell-favoring conditionwith the addition of different concentrations of STAT5 inhibitor for 3days. GM-CSF and IFN-γ expression was analyzed by intracellular cytokinestaining and flow cytometry.

FIGS. 23A-23D depict targeting STAT5 activation by chemical inhibitorameliorates EAE. (FIG. 23A) Clinical EAE scores of wild-type controlmice (n=5) or administrated with STAT5 inhibitor (Calbiochem). Arrowindicates the treatment points. (FIG. 23B) Histology of spinal cords atclay 18 of EAE mice receiving different treatments. (FIG. 23C)Intracellular staining and flow cytometry of CNS-infiltrating CD4⁺ Tcells at peak of disease. (FIG. 23D) Whole CNS was harvest for RNAextraction. GM-CSF mRNA expression was analyzed by RT-PCR. Datarepresent two independent experiments. *p<0.05.

FIGS. 24A-24E depict GM-CSF-producing T_(H) cells are in associationwith human RA. Plasma concentrations of GM-CSF and TNF-α in healthycontrol HC (n=32) and RA (n=47) were quantified by ELISA (FIG. 24A). InFIGS. 24B and 24C, peripheral blood mononuclear cells (PBMCs) werecollected from healthy control (HC) and rheumatoid arthritis (RA)patients, and were stimulated for 4 h with PMA/lonomycin in the presenceof Golgiplug, followed by intracellular cytokine staining.Representative flow cytometry of GM-CSF, IFN-γ and IL-17 in CD4⁺ T cells(FIG. 24B) and statistics of n>=9 per group (FIG. 24C) are shown. FIG.241D shows the correlation between the frequency of GM-CSF⁺IFN-γ T_(H)cells and the level of plasma GM-CSF in peripheral blood of RA patients(n=18). Cytokine expression by CD4⁺ T cells derived from synovial fluidof RA patients was analyzed by intracellular cytokine staining and flowcytometry (FIG. 24E). A representative image of three cases was shown.*p<0.05, **p<0.01, ***p<0.001; ns, not significant.

FIGS. 25A-25E depict distinguishable effects of GM-CSF and TNF-α inmouse AIA. FIG. 25A shows knee joint swelling of wild-type mice over 7days after intraarticular injection of 100 μg mBSA in AIA model,receiving treatment with control IgG, GM-CSF-specific and TNF-α-specificneutralizing antibodies separately or in combination (n=5 per group) atindicated times (arrows). FIG. 25B shows knee joint swelling ofStat5^(+/+) and Stat5^(−/−) mice (n=6 per group) over 7 days afterarthritis induction. Data are representative of more than threeindependent experiments. Representative images of joint sections stainedwith H&E (FIG. 25C) or Safranin-O/Fast Green (FIG. 25D) at day 7 afterarthritis induction as in FIG. 25C. Bars, 500 μm (FIG. 25C upper panelsand FIG. 25D) or 100 μm (FIG. 25C lower panels). Arrow in upper panels(FIG. 25C) indicated hone destruction. In FIG. 25E, serum concentrationsof GM-CSF, IFN-γ and TNF-α in Stat5^(+/+) and Stat5^(−/−) AIA mice werequantified by ELISA. Statistics of n>=8 per group were shown. *p<0.05,**p<0.01, ***p<0.001.

FIGS. 26A-26D depicts mice with Stat5 deletion in T cells are resistantto CIA. (FIG. 26A) Representative images of paw swelling of Stat5^(+/+)and Stat5^(−/−) mice at day 40 after collagen II/CFA immunization in CIAmodel. (FIG. 26B) Clinical score of Stat5^(+/+) and Stat5^(−/−) mice(n=5 per group) over 40 days after collagen II/CFA immunization. Dataare representative of two independent experiments. (FIG. 26C)Representative images of paw sections stained with H&E at clay 40. (FIG.26D) Serum concentrations of TNF-α (n=8 per group) were quantified byELISA. *p<0.05, **p<0.01, ***p<0.001.

FIGS. 27A-27E depicts STAT5-deficient CD4⁺ T cells are defective inarthritogenic potential. (FIGS. 27A and 27B) Representative flowcytometry of CD4⁺ T cells in spleens (FIG. 27A) and inguinal lymph nodes(FIG. 27B) of Stat5^(+/+) and Stat5^(−/−) mice at day 7 after AIAinduction. (FIGS. 27C and 27D) Synovial tissues were harvested fromStat5^(+/+) and Stat5^(−/−) mice at day 7 after AIA induction, anddissociated into single cells. Cell numbers of CD45⁺ leukocytes werecalculated (FIG. 27C). The percentages of CD4⁺ T cells among CD45⁺fraction were analyzed by flow cytometry, and cell numbers werecalculated (FIG. 27D). (FIG. 27E) Histological analysis of jointsections from wild-type naïve mice at day 7 after being transferred within vitro-expanded antigen-reactive CD4⁺ T cells and followed withintraarticular injection of mBSA. Bars, 100 μm. Data represent twoindependent experiments. *p<0.05; ns, not significant.

FIGS. 28A-28G depicts STAT5-regulated GM-CSF-producing T_(H) cells arecrucial for AIA. Spleens and synovial tissues were collected fromStat5^(+/+) and Stat5^(−/−) mice at day 7 after arthritis induction.(FIG. 28A) Splenic fractions of wild-type AIA mice (n=3) were stimulatedunder indicated conditions for 18 h. GM-CSF levels in supernatant werequantified by ELISA. (FIGS. 28B-28D) Intracellular staining and flowcytometry of GM-CSF, IL-17 and IFN-γ in splenic CD4⁺CD44^(hi) effector Tcells (FIG. 28B) or in synovial infiltrating CD4⁺ T cells (FIGS. 28C and28D) after restimulation for 4 h with PMA/Ionomycin in the presence ofGolgiplug. Representative images and statistics of n=5 (FIG. 28B, rightpanels) or n=3 (FIG. 28D, right panels) per group were shown. Datarepresent two independent experiments. (FIG. 28E) Protein expression ofseveral proinflammatory cytokines in synovial tissues was measured byELISA. (FIGS. 28F and 28G) Representative images of joint sectionsstained with H&E (FIG. 28F) or Safranin-O/Fast Green (FIG. 28G) at day 7after intraarticular injection of mBSA alone to the right knee jointsand mBSA supplemented with GM-CSF to the left knee joints. Bars, 500, 50or 200 μm as indicated. Data represent two independentexperiments.*p<0.05, **p<0.01. ***p<0.001; ns, not significant.“Splenocytes” represent the left-most bars in each group, “splenocytesdepleted of CD4+ T cells” represent the middle bars in each group, and“CD4+ T cells” represent the right-most bars in each group.

FIGS. 29A-29C depicts loss of STAT5 results in impaired GM-CSFproduction by antigen-specific CD4⁺ T cells. Spleens and inguinal LNswere collected from Stat5^(+/+) and Stat5^(−/−) mice at day 7 afterarthritis induction, and dissociated into single cell suspensions. Then,cells were stimulated with mBSA (20 μg/ml) for 24 h. (FIG. 29A)Golgiplug was added in the last 4 h of culture, followed byintracellular staining and flow cytometry. Representative plots ofGM-CSF, IL-17 and IFN-γ expression in CD4⁺CD44^(hi) effector T cells wasshown, representing two independent experiments. (FIGS. 29B and 29C)Secreted cytokines in the supernatant (n=3 per group) were quantified byELISA. Data represent two independent experiments. *p<0.05; ns, notsignificant.

FIGS. 30A-30C depicts loss of STAT5 impairs IL-6 and IL-1β expression insynovial tissues of arthritic mice. (FIGS. 30A-30C) The mRNA (FIGS. 30Aand 30 C) and protein (FIG. 30B) expression of several proinflammatorycytokines in synovial tissues of Stat5^(+/+) and Stat5^(−/−) mice (n>=3per group) at day 5 or 7 after arthritis induction was measured by qPCRand ELISA. The qPCR data were normalized to Rn18S.

FIGS. 31A-31D depicts SAT5-induced GM-CSF expression mediates CD11b⁺cell accumulation in inflamed synovial tissues. (FIG. 31A) Thefrequencies of CD11b⁺ cells in spleens of Stat5^(+/+) and Stat5^(−/−)AIA mice were analyzed by flow cytometry. Statistics of n=3 per group(right panel) were shown. (FIG. 31B) Synovial tissues were harvestedfrom Stat5^(+/+) and Stat5^(−/−) mice at day 7 after arthritisinduction, and dissociated into single cell suspensions. The percentageof CD11b⁺ myeloid cells among CD45⁺ fraction was analyzed by flowcytometry. Statistics of n=5 per group were shown in right panel. (FIG.31C) Representative flow cytometry of CD11b⁺ and CD4⁺ cells gated onsynovial CD45⁺ fraction over 7 days after arthritis induction. (FIG.31D) Flow cytometric analysis of CD4⁺, CD11b⁺ and B220⁺ cellinfiltration in synovial tissues of Stat5^(+/+) and Stat5^(−/−) mice atday 7 after intraarticular injection of mBSA alone to the right kneejoints and mBSA supplemented with GM-CSF to the left knee joints.Representative images were shown. All data shown are representative oftwo independent experiments. **p<0.01; ns, not significant.

FIGS. 32A-32D depicts GM-CSF mediates neutrophil accumulation inarthritic mice. (FIG. 32A) Flow cytometric analysis of Ly6C and Ly6Gexpression gated on synovial CD45⁺CD11b⁺ fraction over 7 days afterarthritis induction. (FIG. 32B) Giemsa stain of sorted Ly6C^(hi)Ly6G⁻and Ly6C^(lo)Ly6G^(hi) cells from synovial tissues of AIA mice. Scalebar, 100 μm (left) or 20 μm (right). (FIG. 32C) Flow cytometric analysisof Ly6C^(hi)Ly6G⁻ and Ly6C^(lo)Ly6G^(hi) populations in synovial tissuesof Stat5^(+/+) and Stat5^(−/−) mice at day 7 after intraarticularinjection of mBSA alone to the right knee joints and mBSA supplementedwith GM-CSF to the left knee joints. (FIG. 32D) Knee joint swelling ofwild-type mice treated with Ly6G-specific neutralizing antibody (1A8) orIgG control (n=5 per group) over 3 days after intraarticular injectionof mBSA in AIA model. Arrows indicate time points of antibodyadministration. *p<0.05.

FIGS. 33A-33C depicts GM-CSF enhances neutrophil transmigration anddelay apoptosis in vitro. (FIG. 33A) Percentages of migrated neutrophilswith or without GM-CSF as chemoattractant in transmigration assay at 3 hpost start. (FIG. 33B) Microscopic images of CFSE-labeled neutrophils inthe bottom of the lower chamber in transmigration assay. (FIG. 33C)Sorted neutrophils were cultured in vitro with or without GM-CSF (20ng/ml) for 24 h. Neutrophils undergoing apoptosis were examined byAnnexin V and propidium iodide (PI) co-staining. A representative flowcytometry of three repeats was shown. *p<0.05.

FIGS. 34A-34I depicts GM-CSF mediates proinflammatory cytokineexpression by myeloid cells and synovial fibroblasts in arthritic mice.Synovial tissues were dissected from wild-type AIA mice and dissociatedinto single cell suspensions. (FIG. 34A) Flow cytometry plots depictingthe fractionation into different populations based on differentialexpression of surface markers. (FIG. 34B) The mRNA expression of severalproinflammatory cytokines in sorted CD45⁺TCRβ⁺ (TCRβ⁺ in short).CD45⁺TCRβ⁻CD11c⁻CD11b⁺ (CD11b⁺) and CD45⁺TCRβ⁻CD11c⁺ (CD11c⁺)populations was measured by qPCR. The qPCR data were normalized toGAPDH. (FIG. 34C) The mRNA expression of IL-6, IL-1β and TNF-α in sortedLy6C^(hi)Ly6G⁻ and Ly6C^(lo)Ly6G^(hi) populations (gated on CD11b⁺cells). The qPCR data were normalized to GAPDH. (FIGS. 34D and 34E) ThemRNA expression of IL-6 and IL-1β by BMDMs (FIG. 34D) and BMDCs (FIG.34E) upon stimulation with 20 ng/ml GM-CSF for 1 h. The qPCR data werenormalized to GAPDH. (FIGS. 34F and 34G) BMDMs (FIG. 34F) and BMDCs(FIG. 34G) were stimulated with GM-CSF at indicated concentrations (n=3per group) for 18 h. The secretion of IL-6 in the culture supernatantwas quantified by ELISA. (FIG. 34H) BMDMs were primed with LPS (100μg/ml) in the presence of GM-CSF at indicated concentrations (n=3 pergroup) for 6 h, followed by stimulation with ATP (5 mM) for 30 min. Thesecretion of IL-1β in the culture supernatant was quantified by ELISA.(FIG. 34I) Cells were cultured in DMEM medium supplemented with 10% PBSfor over 20 days with more than 5 passages to obtain synovialfibroblasts. Synovial fibroblasts were stimulated with GM-CSF (20 ng/ml)for 1 h and harvested for RNA extraction. The mRNA expression of IL-1βwas measured by qPCR. The qPCR data were normalized to GAPDH. All datashown represent two independent experiments.*p<0.05, **p<0.01.

DETAILED DESCRIPTION OF THE INVENTION

A description of example embodiments of the invention follows.

The present disclosure relates, in part, to the identification of agranulocyte macrophage colony stimulating factor (GM-CSF)-secreting Thelper cell, termed “T_(H)-GM”. As detailed herein. IL-7/STAT5 signalingprograms the differentiation of precursor CD4+ cells to T_(H)-GM, aprocess which is further modulated by IL-2 and IL-23 signaling. T_(H)-GMcells are characterized by, e.g., GM-CSF and IL-3 production. T_(H)-GMcells are distinct from the known helper T cells T_(H)1 and T_(H)17,with respect to, e.g., differentiation conditions, transcriptionalregulation and effector cytokine expression. For example, IL-12/IFN-γand TGF-0/IL-6, which mediate (e.g., promote the development of) T_(H)1and T_(H)17, respectively, potently suppress the development of T_(H)-GMfrom naïve CD4⁺ precursor cells, establishing that T_(H)-GM cellsdevelop via a lineage distinct from T_(H)1 and T_(H)17. Thus, thepresent disclosure provides a distinct network of factors, unique fromfactors known to mediate T_(H)1 or T_(H)17, that mediate T_(H)-GMfunction (e.g., its differentiation and pathogenicity).

As shown herein, T_(H)-GM cells preferentially induce EAE as comparedwith T_(H)1 and T_(H)17 cells, indicating that T_(H)-GM cells representthe primary effectors in the pathogenesis of autoimmuneneuroinflammation in humans. Moreover, blockade of IL-7 signaling and/orinhibition of STAT5 function (e.g., abrogation of expression orinhibition of STAT5 activity) attenuates autoimmune neuroinflammationassociated with diminished GM-CSF production by T_(H)-GM cells. Further,blockade of T_(H)-GM cell-secreted GM-CSF ameliorates experimentalarthritis in a TNT-α-independent manner, indicating an approach for thetreatment of, e.g., rheumatoid arthritis patients who are unresponsiveto TNF-α antagonistic drugs. Thus, the present disclosure enables one todistinguish between an inflammatory disorder (e.g., RA) that is mediatedby the T_(H)-GM pathway (e.g., a disorder that results from T_(H)-GMpathogenicity through the action of, e.g., GM-CSF and/or IL-3, or anyfactor associated with the T_(H)-GM pathway), or an inflammatorydisorder that is mediated by, e.g., TNF-α, IL-6, and/or IL-1β pathways(i.e., non-T_(H)-GM-mediated pathway). For example, a patient who has,e.g., RA may be afflicted with a type of RA that is primarilyT_(H)-GM-mediated, or primarily non-T_(H)-GM-mediated (e.g.,TNF-α-mediated or IL-6 mediated). The present disclosure enables theclassification between T_(H)-GM-mediated and non-T_(H)-GM-mediatedinflammation, allowing for a more precise diagnosis, prognosis, andtreatment in an individual who is afflicted with an inflammatorydisorder such as RA or MS.

As demonstrated herein, the present disclosure identifies a helper Tcell subset (T_(H)-GM), provides the molecular basis for the commitmentand development of this subset from naïve precursor cells in vitro andin vivo, and demonstrates T_(H)-GM cells as the primary pathogenic cellsin autoimmune diseases and inflammatory disorders, for example, MS andRA. Thus, provided herein are compositions and methods for diagnosinginflammatory conditions primarily mediated by T_(H)-GM cells, therebyenabling the identification of, e.g., RA patients who are non-responsiveto TNF-α therapy (e.g., TNF-α inhibitor based therapy), as well ascompositions and methods for modulating T_(H)-GM function to treatautoimmune and inflammatory disorders. The methods of modulatingT_(H)-GM function include, e.g., administering agents to modulate thefunction (e.g., signaling, expression or activity) of the network offactors (e.g., IL-2/IL-7/STAT5/GM-CSF/IL-3) that mediate T_(H)-GMfunction in an effective amount to modulate the function (e.g.,development and pathogenicity) of T_(H)-GM cells. In particular, thedisclosure provides methods and composition for differentiating anddiagnosing an inflammatory disorder, e.g., multiple sclerosis (MS),rheumatoid arthritis (RA) as primarily mediated by either T_(H)-GM cells(i.e. T_(H)-GM pathway mediated) or by non-T_(H)-GM mechanism (e.g.,TNF-α, IL-6, and/or IL-1β pathways), or both. Also provided herein arecompositions and methods for and the treatment of inflammatorydisorders, particularly those that are T_(H)-GM-mediated.

Accordingly, in one aspect, the present disclosure provides a method ofdiagnosing a T_(H)-GM-mediated inflammatory disorder in a patientsuffering from an inflammatory disorder. In some embodiments, the methodcomprises contacting a sample collected from a patient suffering from aninflammatory disorder with a detecting agent that detects a polypeptideor nucleic acid level of a T_(H)-GM-mediating factor, such as, e.g.,STAT5, IL-7, GM-CSF or IL-3, or a combination thereof; and quantifyingthe polypeptide or nucleic acid level of the T_(H)-GM-mediating factor(e.g., STAT5, IL-7, GM-CSF or IL-3, or a combination thereof), whereinan increased level of a T_(H)-GM-mediating factor (e.g., STAT5, IL-7,GM-CSF or IL-3, or a combination thereof) relative to a reference levelindicates that the patient suffers from a T_(H)-GM-mediated inflammatorydisorder, thereby diagnosing a T_(H)-GM-mediated inflammatory disorderin a patient suffering from an inflammatory disorder.

As used herein, a “T_(H)-GM-mediated” inflammatory disorder refers to asubtype of an inflammatory disorder (e.g., a subtype of RA or MS) thatresults from the physiological action of any one or more of the networkof factors in the pathway that modulate T_(H)-GM function (a“T_(H)-GM-mediating factor”), as described herein. Such factors include,e.g., GM-CSF, activated STAT5, IL-7, IL-2, and IL-3. In a particularembodiment, STAT5 is activated STAT5, wherein tyrosine at position 694is phosphorylated.

In some embodiments, the level of a T_(H)-GM-mediating factor (e.g.,STAT5, IL-7, GM-CSF or IL-3, or a combination thereof) that is notincreased relative to a reference level indicates that the patientsuffers from a non-T_(H)-GM-mediated inflammatory disorder.

In certain embodiments, the method further comprises administering tothe patient a TNF-α therapy, as described herein, if the level of aT_(H)-GM-mediating factor (e.g. STAT5, IL-7, GM-CSF or IL-3, or acombination thereof) is not increased relative to a reference level.

As used herein, a “non-T_(H)-GM-mediated” inflammatory disorder refersto an inflammatory disorder (e.g., RA or MS) that is primarily causedby, e.g., TNF-α, IL-6, or IL-1β (and/or factors in the TNF-α, IL-6, orIL-1β pathway). As such, a “T_(H)-GM-mediated” inflammatory disorderresults primarily (or exclusively) from a pathway that is distinct fromone or more of the pathways that leads to a “non-T_(H)-GM-mediated”inflammatory disorder (e.g., the pathways associated with TNF-α, IL-6,or IL-1β).

However, as those of skill in the art would appreciate, aT_(H)-GM-mediated inflammatory disorder does not necessarily exclude thepossibility that the inflammatory disorder could also be partiallynon-T_(H)-GM-mediated (e.g., mediated by TNF-α, IL-6, or IL- and/orfactors in the TNF-α, IL-6, or IL-1β pathway). Thus, a classification ordiagnosis as “T_(H)-GM-mediated” is synonymous with“primarily/predominantly T_(H)-GM-mediated”, and a classification as“non-T_(H)-GM-mediated” is synonymous with “primarily/predominantlynon-T_(H)-GM-mediated.” For example, without wishing to be bound by anyparticular theory, an inflammatory disorder in its early stage may beT_(H)-GM-mediated. As the inflammatory condition advances to a latestage characterized by, e.g., tissue damage, the inflammatory disorderbecomes progressively non-T_(H)-GM-mediated. In some embodiments, aT_(H)-GM-mediated inflammatory disorder is a condition that isresponsive to modulation of T_(H)-GM function, as determined by clinicalstandards; a non-T_(H)-GM-mediated inflammatory disorder is a conditionthat is responsive to, e.g., TNF-α, IL-6, or IL-1β therapy, asdetermined by clinical standards. In certain embodiments, aninflammatory disorder can be responsive to modulation of T_(H)-GMfunction as well as TNF-α, IL-6, and/or IL-1β therapy.

In some embodiments, the sample can be e.g., peripheral blood,cerebrospinal fluid, synovial fluid, or synovial membrane, or acombination thereof.

In some embodiments, the inflammatory disorder is an autoimmunedisorder. In certain embodiments, the inflammatory disorder can be anyinflammatory disorder mediated by T_(H)-GM cells, and includes, but isnot limited to rheumatoid arthritis, multiple sclerosis, ankylosingspondylitis, Crohn's disease, diabetes. Hashimoto's thyroiditis,hyperthyroidism, hypothyroidism, Irritable Bowel Syndrome (IBS), lupuserythematosus, polymyalgia rheumatic, psoriasis, psoriatic arthritis,Raynaud's syndrome/phenomenon, reactive arthritis (Reiter syndrome),sarcoidosis, scleroderma, Sjögren's syndrome, ulcerative colitis,uveitis, or vasculitis.

As used herein, a “detecting agent” refers to, e.g., an antibody, apeptide, a small molecule, or a nucleic acid that binds to a polypeptideor nucleic acid to be detected (e.g., STAT5 (e.g., phospho-STAT5(Tyr694)), IL-7, GM-CSF or IL-3), and enables the quantification of thepolypeptide or nucleic acid to be detected. The detecting agent can bedetectably labeled, or quantifiable by other means known in the art.

In some embodiments, the detecting agent is an antibody that binds tothe polypeptide of STAT5, IL-7, GM-CSF or IL-3. In one embodiment, theantibody is one that binds to an activated STAT5 (e.g., phosphorylatedSTAT5), as described herein. Antibodies to STAT5 (e.g., phospho-STAT5(Tyr694)), IL-7, GM-CSF or IL-3 suitable for use in the present methodare known and commercially available in the art (e.g., STAT5 Ab: C-17from Santa Cruz Biotech; Phospho-STAT5 (Tyr694) Ab: #9351 or #9359 fromCell Signaling; IL-7 Ab: clone BVD10-40F6 from BD Pharmingen; IL-7R Ab:clone SB/14 from BD Pharmingen; GM-CSF Ab: clone MP1-22E9 from BDPharmingen; IL-3 Ab: clone MP2-8F8 from BD Pharmingen.

In other embodiments, the detecting agent is a nucleic acid that hindsto the nucleic acid of STAT5. IL-7, GM-CSF and/or IL-3. Nucleic acidmolecules encoding a, e.g., STAT5. IL-7, GM-CSF and/or IL-3 sequence, orfragments or oligonucleotides thereof, that hybridize to a nucleic acidmolecule encoding a e.g., STAT5, IL-7, GM-CSF and/or IL-3 polypeptidesequence at high stringency may be used as a probe to monitor expressionof nucleic acid levels of STAT5, IL-7, GM-CSF and/or IL-3 in a samplefor use in the diagnostic methods of the disclosure. Methods ofquantifying nucleic acid levels are routine and available in the art.

In some embodiments, the method further comprises contacting the samplewith a detecting agent that detects a polypeptide or nucleic acid levelof one or more genes (as well as the gene product) listed in Table 1. Asdescribed herein, Table 1 lists genes that are differentially expressedin T_(H)-GM cells as well as genes that are differentially expressed onthe surface of T_(H)-GM cells, as compared to T_(H)1 or T_(H)17 cells.

In a particular embodiment, the method further comprises contacting thesample with a detecting agent that detects the polypeptide or nucleicacid level of basic helix-loop-helix family member e40 (BHLHe40),chemokine (C-C Motif) Receptor 4 (CCR4), and/or CCR6.

Standard methods may be used to quantify polypeptide levels in anysample. Such methods include, e.g., ELISA, Western blotting,immunohistochemistry, fluorescence activated cells sorting (FACS) usingantibodies directed to a polypeptide, and quantitative enzymeimmunoassay techniques known in the art. Such methods are routine andavailable in the art. Similarly, methods for quantifying nucleic acidlevels (e.g., mRNA) are known in the art.

In the diagnostic method of the present disclosure, an increased levelof STAT5 (e.g., activated phospho-STAT5 (Tyr694)), IL-7, GM-CSF and/orIL-3 relative to a reference level indicates that the patient suffersfrom a T_(H)-GM-mediated inflammatory disorder.

In some embodiments, a STAT5 (e.g., activated phospho-STAT5 (Tyr694)),IL-7, GM-CSF and/or IL-3 level that is increased by at least 10%, atleast 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 100%, at least 110%, atleast 120%, at least 130%, at least 140%, at least 150%, at least 160%,at least 170%, at least 180%, at least 190%, at least 200%, at least220%, at least 240%, at least 260%, at least 280%, at least 300%, atleast 350%, at least 400%, at least 450%, at least 500%, at least 550%,or at least 600% relative to a reference level indicates that thepatient suffers from a T_(H)-GM-mediated inflammatory disorder. In aparticular embodiment, an increase of at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 100%, orat least 150% relative to a reference level indicates that the patientsuffers from a T_(H)-GM-mediated inflammatory disorder.

In some embodiments, a STAT5 (e.g., activated phospho-STAT5 (Tyr694)),IL-7, GM-CSF and/or IL-3 level that is not increased by at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 100%, or at least 150% relative to a reference level indicatesthat the patient suffers from a non-T_(H)-GM-mediated inflammatorydisorder.

In certain embodiments, a STAT5 (e.g., activated phospho-STAT5(Tyr694)), IL-7, GM-CSF and/or IL-3 level that is comparable (orunchanged) relative to a reference level indicates that the patientsuffers from a non-T_(H)-GM-mediated disorder. As used herein, a levelthat is “comparable” to that of a reference level refers to a level thatis unchanged, or a change relative to the reference level that isstatistically insignificant according to clinical standards. In certainembodiments, a comparable level (or unchanged level) can include a levelthat is not increased by at least 40%, at least 50%, at least 60%, or atleast 70% relative to a reference level as, for example, it may notindicate a clinically significant change. In some embodiments, a levelof a T_(H)-GM-mediating factor (e.g., STAT5 (e.g., activatedphospho-STAT5 (Tyr694)). IL-7, GM-CSF, and/or IL-3) that is decreasedrelative to a reference level can also indicate that the patient suffersfrom a non-T_(H)-GM-mediated disorder.

In some embodiments, the reference level is a level that is used forcomparison purposes, and may be obtained from, for example, a priorsample taken from the same patient; a normal healthy subject; a samplefrom a subject not having an autoimmune disease or an inflammatorydisorder; a subject that is diagnosed with a propensity to develop anautoimmune disease but does not yet show symptoms of the disease; apatient that has been treated for an autoimmune disease; or a sample ofa purified reference polypeptide or nucleic acid molecule of thedisclosure (e.g., STAT5) at a known normal concentration. By “referencestandard or level” is meant a value or number derived from a referencesample, or a value or range accepted in the art as indicative of beinghealthy (e.g., an individual that does not have an inflammatorydisorder). A normal reference standard or level can also be a value ornumber derived from a normal subject who does not have an autoimmunedisease. In one embodiment, the reference sample, standard, or level ismatched to the sample subject by at least one of the following criteria;age, weight, body mass index (BMI), disease stage, and overall health. Astandard curve of levels of purified DNA. RNA or mRNA within the normalreference range can also be used as a reference. A standard curve oflevels of purified protein within the normal reference range can also beused as a reference.

In some embodiments, the patient afflicted with an inflammatory disorderwho has been diagnosed or classified as having a T_(H)-GM-mediatedinflammatory disorder does not have a non-T_(H)-GM-mediated inflammatorydisorder (i.e., does not have a TNF-α, IL-6, or IL-1β-mediatedinflammatory disorder). That is, the patient diagnosed as suffering froma T_(H)-GM-mediated inflammatory disorder responds to modulation ofT_(H)-GM function (e.g., inhibition of STAT5, IL-7, GM-CSF and/or IL-3),but does not respond (or exhibits a limited response) to TNF-α therapy,as determined by clinical standards. However, as described herein, aT_(H)-GM-mediated inflammatory disorder does not exclude the possibilitythat the inflammatory disorder is also partially (though not primarily)contributed by a non-T_(H)-GM-mediated pathway (e.g., TNF-α, IL-6,IL-1β).

In some embodiments, the methods of the present disclosure furthercomprise administering an effective amount of a modulating agent thatmodulates T_(H)-GM cell function to the patient diagnosed or classifiedas having a T_(H)-GM-mediated inflammatory disorder. As describedherein, in some embodiments, the modulating agent inhibits T_(H)-GMfunction.

In some embodiments, the methods of the present disclosure furthercomprise administering an effective amount of, e.g., a TNF-α therapy, anIL-6 therapy, or an IL-1β therapy to a patient diagnosed or classifiedas having a non-T_(H)-GM-mediated inflammatory disorder, as describedherein.

In some aspects, the present disclosure also provides a method ofclassifying a patient suffering from an inflammatory disorder as havinga T_(H)-GM-mediated inflammatory disorder or a non-T_(H)-GM-mediatedinflammatory disorder. In some embodiments, the method comprisescontacting a sample collected from a patient suffering from aninflammatory disorder with a detecting agent that detects a polypeptideor nucleic acid level of a T_(H)-GM-mediating factor, such as, e.g.,STAT5 (e.g., phosphorylated STAT5, Tyr694), IL-7, GM-CSF or IL-3, or acombination thereof. In certain aspects, the method further comprisesquantifying the polypeptide or nucleic acid level of theT_(H)-GM-mediating factor, such as, e.g., STAT5. IL-7, GM-CSF or IL-3,or a combination thereof, wherein an increased level of theT_(H)-GM-mediating factor, such as, e.g., STAT5, IL-7, GM-CSF or IL-3,or a combination thereof relative to a reference level indicates thatthe patient suffers from a T_(H)-GM-mediated inflammatory disorder; or acomparable level of the T_(H)-GM-mediating factor, such as, e.g., STAT5,IL-7, GM-CSF or IL-3, or a combination thereof relative to a referencelevel indicates that the patient suffers from a non-T_(H)-GM-mediatedinflammatory disorder, thereby classifying the patient suffering from aninflammatory disorder as a T_(H)-GM-mediated inflammatory disorder or anon-T_(H)-GM-mediated inflammatory disorder.

In other aspects of the present disclosure, the methods disclosed hereincan further comprise measuring the polypeptide or nucleic acid level ofa factor that mediates a non-T_(H)-GM-mediated inflammatory disorder.Such factors include, e.g., TNF-α, IL-6, and IL-1β.

For example, in some aspects, the present disclosure provides a methodof determining a treatment regimen in a patient suffering from aninflammatory disorder. To illustrate, the method comprises quantifying apolypeptide or nucleic acid level of, e.g., activated STAT5 or GM-CSF ina sample collected from a patient suffering from an inflammatorydisorder, and quantifying the polypeptide or nucleic acid level of,e.g., TNF-α in a sample collected from the patient. At least fourscenarios can be considered.

In the first scenario, if the activated STAT5 or GM-CSF level isincreased (e.g., by at least 40%, at least 50%, at least 60%, at least70%, at least 80%, at least 90%, at least 100%, or at least 150%)relative to a first reference level and the TNF-α level is comparable toa second reference level, then the patient is classified as having aT_(H)-GM-mediated inflammatory disorder and the patient can be treatedwith an agent that modulates T_(H)-GM function, as described herein.

In a second scenario, if the activated STAT5 or GM-CSF level iscomparable to the first reference level and the TNF-α level is increased(e.g., by at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 100%, or at least 150%) relative tothe second reference level, then the patient is classified as having anon-T_(H)-GM-mediated inflammatory disorder and the patient can betreated with, e.g., a TNF-α therapy.

In a third scenario, if the activated STAT5 or GM-CSF level is increasedrelative to the first reference level and the TNF-α level is alsoincreased relative to the second reference level, and the increase isequivalent within clinical and/or statistical standards (e.g., bothGM-CSF and TNF-α are at least 50% increased relative to the respectivereference levels), then the patient is classified as having aninflammatory disorder that is equally T_(H)-GM-mediated and non-T_(H)-GMmediated (e.g., TNF-α-mediated). In such a case, the patient can betreated with an effective amount of an agent that modulates T_(H)-GMfunction and an effective amount of, e.g., a TNF-α therapy. Asdemonstrated herein, the combination of both agents can have asynergistic effect.

In a fourth scenario, if the activated STAT5 or GM-CSF level isincreased relative to the first reference level and the TNF-α level isalso increased relative to the second reference level, but one isincreased more than the other, then the inflammatory disorder isprimarily mediated by the pathway that shows a greater increase. Forexample, if GM-CSF is increased by 40% relative to a reference level,and TNF-α is increased by 90% relative to a reference level, then theinflammatory disorder is primarily non-T_(H)-GM-mediated. However, inthis scenario, the patient may receive a combined treatment with anagent that modulates T_(H)-GM function as well a TNF-α therapy (e.g.,anti-TNF-α therapy), since GM-CSF is increased by, e.g., at least 40%relative to a reference level.

In some embodiments, the first and second reference levels are obtainedfrom the same reference sample.

In a related aspect, the disclosure also provides a method of tailoringthe treatment of a patient suffering from an inflammatory disorderaccording to the progression of a patient's inflammatory disorder. Inthe above illustrative example, the first scenario (increasedT_(H)-GM-mediating factor, e.g. STAT5 or GM-CSF but TNF-α level iscomparable to a reference level) may indicate that the patient is in anearly stage of an inflammatory disorder. Without wishing to be bound byany particular theory, during, for example, the early stages of aninflammatory disorder, naïve T cells are stimulated by antigen andprogrammed by IL-7/STAT5 to differentiate into GM-CSF/IL-3 producingT_(H)-GM cells. During, for example, the late stages of an inflammatorydisorder, T_(H)-GM cytokines (e.g., IL-3 and GM-CSF) progressivelystimulate more inflammatory cells such as macrophages and neutrophilsresulting in the production of, e.g., TNF-α, IL-6, IL-1β, resulting infull-scale inflammation. Thus, in the above illustrative example, thesecond scenario (activated STAT5 Or GM-CSF level is comparable to thefirst reference level and the TNF-α level is increased) may indicatethat the patient is in a late stage of an inflammatory disordercharacterized by, e.g., tissue damage. Accordingly, the presentdisclosure enables the prognosis of a patient depending on thequantifiable level of one or more T_(H)-GM-mediating factor (e.g., STAT5(e.g., activated phospho-STAT5 (Tyr694)), IL-7, GM-CSF, and/or IL-3) andone or more non-T_(H)-GM-mediating factor (e.g., TNF-α, IL-6, IL-1β),thereby tailoring the treatment according to the progression of thedisease. Accordingly, as would be appreciated by those of skill in theart, a patient suffering from an inflammatory disorder can be monitoredfor disease progression to ensure effective and tailored treatmentaccording to the level of one or more T_(H)-GM-mediating factor, asdescribed herein, and one or more non-T_(H)-GM-mediating factor (e.g.,TNF-α, IL-6, IL-1β).

In related aspects, the present disclosure also provides a method ofprognosing progression of an inflammatory disorder in a patient in needthereof. In some embodiments, the method comprises a) quantifying apolypeptide or nucleic acid level of a T_(H)-GM-mediating factor, suchas, e.g., STAT5, IL-7, GM-CSF or IL-3, or a combination thereof, in afirst sample collected from a patient suffering from an inflammatorydisorder, and b) quantifying a polypeptide or nucleic acid level of,e.g., TNF-α, IL-6, or IL-1β, or a combination thereof, in a secondsample collected from the patient, wherein i) an increased level of theT_(H)-GM-mediating factor, such as, e.g., STAT5, IL-7, GM-CSF or IL-3,or a combination thereof relative to a first reference level and anunchanged level of TNF-α, IL-6, or IL-1β, or a combination thereofrelative to a second reference level indicates that the patient is in anearly stage of the inflammatory disorder, as described herein; or ii) anunchanged level of the T_(H)-GM-mediating factor, such as, e.g., STAT5,IL-7, GM-CSF or IL-3, or a combination thereof relative to the firstreference level and an increased level of TNF-α, IL-6, or IL-43, or acombination thereof relative to the second reference level indicatesthat the patient is in a late stage of the inflammatory disorder, asdescribed herein. In some embodiments, the method further comprisesadministering an effective amount of an agent that modulates T_(H)-GMfunction and/or, e.g., a TNF-α therapy, as described herein.

In some embodiments, the first sample and the second sample are thesame.

In various aspects, the present disclosure also provides an isolatedpopulation of GM-CSF-secreting T-helper cells (T_(H)-GM). In oneembodiment, the T_(H)-GM cells are differentiated from a precursor cell(e.g., CD4+ cells) in the presence of signal transducer and activator oftranscription 5 (STAT5) and/or IL-7, and wherein the T_(H)-GM cellsexpress GM-CSF and IL-3.

In some embodiments, the T_(H)-GM cells are differentiated from aprecursor cell (e.g., CD4+ cells) in the presence of an agent thatinhibits IL-12, IFN-γ. TGF-β, and/or IL-6. Similarly, thedifferentiation of a precursor cell (e.g., CD4+ precursor cell) into aT_(H)-GM cell is inhibited by IL-12, IFN-γ, TFG-β, and/or IL-6.

In some embodiments, the T_(H)-GM cells are differentiated from aprecursor cell in vitro, under artificial conditions, but wherein theT_(H)-GM cells retain physiological properties as described herein.

In some embodiments, the T_(H)-GM cells are further characterized by anoverexpression of one or more genes listed in Table 1. For example, theT_(H)-GM cells are further characterized by an overexpression of, forexample, basic helix-loop-helix family, member e40 (BHLHe40),preproenkephalin (PENK), IL-2, serine (or cysteine) peptidase inhibitor,Glade B member 6 h (Serpinb6b), neuritin 1 (Nrn1), stearoyl-Coenzyme Adesaturase 1 (Scd1), or phosphotriesterase related C1q-like 3 (Pter), ora combination thereof.

In some embodiments, the T_(H)-GM cells are further characterized by anunderexpression of one or more genes listed in Table 1. For example, theT_(H)-GM cells are further characterized an underexpression oflymphocyte antigen 6 complex, locus A (Ly6a); CD27; or selectin,lymphocyte (Sell).

As described herein, the identification of a distinct network of factors(unique from factors known to mediate T_(H)1 or T_(H)17) that mediateT_(H)-GM function (e.g., its differentiation and pathogenicity) enablestargeted modulation of T_(H)-GM function to treat T_(H)-GM-mediateddisorders, e.g., disorders that result from aberrant T_(H)-GM function.Thus, in some aspects, the present disclosure provides a method ofmodulating T_(H)-GM function, comprising contacting the T_(H)-GM, orcluster of differentiation 4 (CD4+) precursor cells, or both, with amodulating agent that modulates T_(H)-GM function. In one embodiment,the modulating agent is contacted with the T_(H)-GM cells or CD4+precursor cells in vitro or in VIVO.

As used herein, “T_(H)-GM function” refers to the commitment,development, maintenance, survival, proliferation, or activity, or acombination thereof, of T_(H)-GM cells. Thus, an agent that modulates(e.g., enhances or inhibits) T_(H)-GM function is one that modulatesT_(H)-GM commitment, development, survival, proliferation, or activity,or combination thereof, of T_(H)-GM cells. For example, T_(H)-GMfunction can be modulated by modulating its: commitment from a CD4⁺precursor T cell; development of a CD4⁺ precursor cell that has beencommitted to the T_(H)-GM developmental pathway; maintenance of aT_(H)-GM phenotype; survival or proliferation under development oreffector T_(H)-GM cells; and/or activity of effector T_(H)-GM cells(e.g., modulating function of a secreted factor such as GM-CSF or IL-3).For example, a modulation in T_(H)-GM function includes, but is notlimited to, a modulation in: the number of T_(H)-GM cells; the survivalof T_(H)-GM cells; the proliferation of T_(H)-GM cells; and/or theactivity of T_(H)-GM cells. The activity of T_(H)-GM cells hereinincludes the activity induced by the cytokines, chemokines, growthfactors, enzymes and other factors secreted by T_(H)-GM cells, asdescribed herein, and the activity induced by direct contact withT_(H)-GM cells.

As used herein, a T helper subset cell “T_(H)-GM” refers to a cell that,similar to T_(H)1 and T_(H)17 cells, differentiates from precursor CD4+precursor cells, but which commits and develops through a pathway thatis mediated by a subset of factors (the T_(H)-GM-mediating factors) thatis distinct and unique from the known subset of factors that commit anddevelop T_(H)1 or T_(H)17 cell subtypes, as described herein. In someembodiments, a T_(H)-GM cell produces a distinct and unique set of genes(see, e.g., Table 1) and effects pathogenicity through a differentmechanism and pathway than the known factors that mediate pathogenicityof T_(H)1 or T_(H)17 cell subtypes. For example, a T_(H)-GM cell commitsand develops by IL-7/STAT5 function (its regulators), and effectspathogenicity by GM-CSF/IL-3 (its effectors).

In some aspects, the present disclosure provides a method of treating aT_(H)-GM-mediated inflammatory disorder in a patient in need thereof,comprising administering to said patient an effective amount of amodulating agent that modulates T_(H)-GM cell function. In certainembodiments, the patient is previously diagnosed as having aT_(H)-GM-mediated inflammatory disorder, as described herein.

In some aspects, the present disclosure also provides a method oftreating rheumatoid arthritis in a patient who exhibits limited responseto TNF-α therapy, comprising administering to said patient an effectiveamount of a modulating agent that modulates T_(H)-GM function.

As used herein, “limited response” refers to no response orinsignificant response such that a patient is not treated by thetherapy, as determined by clinical standards.

“Treatment” or “treating” refers to therapy, prevention and prophylaxisand particularly refers to the administration of medicine or theperformance of medical procedures with respect to a patient, for eitherprophylaxis (prevention) or to reduce the extent of or likelihood ofoccurrence of the condition or event in the instance where the patientis afflicted. It also refers to reduction in the severity of one or moresymptoms associated with the disease or condition. In the presentapplication, it may refer to amelioration of one or more of thefollowing: pain, swelling, redness or inflammation associated with aninflammatory condition or an autoimmune disease. As used herein, and aswell-understood in the art, “treatment” is an approach for obtainingbeneficial or desired results, including clinical results. For purposesof this disclosure, beneficial or desired clinical results include, butare not limited to, alleviation or amelioration of one or more symptoms,diminishment of extent of disease, stabilized (e.g., not worsening)state of disease, delay or slowing of disease progression, and/oramelioration or palliation of the disease state. “Treatment” can alsomean prolonging survival as compared to expected survival if notreceiving treatment.

An “effective amount” of an agent is that amount sufficient to effectbeneficial or desired results, including clinical results. An “effectiveamount” depends upon the context in which it is being applied. In thecontext of administering a composition that modulates an autoimmuneresponse, an effective amount of an agent which is a modulator ofT_(H)-GM function is an amount sufficient to achieve such a modulationas compared to the response obtained when there is no agentadministered. An effective amount can confer immediate, short term orlong term benefits of disease modification, such as suppression and/orinhibition of T_(H)-GM function, as defined herein. An effective amountcan be administered in one or more administrations. An “effectiveamount” as used herein, is intended to mean an amount sufficient toreduce by at least 10%, at least 25%, at least 50%, at least 75%, or anamount that is sufficient to cause an improvement in one or moreclinically significant symptoms in the patient.

In some embodiments, the modulating agent inhibits T_(H)-GM function to,e.g., reduce inflammation. The inhibition conferred by the modulatingagent (the inhibitor) does not imply a specific mechanism of biologicalaction. Indeed, the term “antagonist” or “inhibitor” as used hereinincludes all possible pharmacological, physiological, and biochemicalinteractions with factors that mediate T_(H)-GM function (e.g., IL-7,IL-7 receptor, STAT5, GM-CSF, IL-3, IL-2, IL-2 receptor, PENK, RANKL,JAK1/3, or any of the genes that are differentially expressed inT_(H)-GM cells, e.g., genes in Tables 1 and 2), whether direct orindirect, and includes interaction with a factor (or its activefragment) that mediates T_(H)-GM function at the protein and/or nucleicacid level, or through another mechanism.

In certain embodiments, a modulating agent that inhibits T_(H)-GMfunction includes an antibody, a polypeptide (e.g., a soluble receptorthat hinds and inhibits, for example, IL-7), a small molecule, a nucleicacid (e.g., antisense, small interfering RNA molecule, short hairpinRNA, microRNA), or a protein (e.g., cytokine), or a combination thereofthat prevents the function (e.g., expression and/or activity) of afactor that mediates T_(H)-GM function. Methods of designing, producing,and using such inhibitors are known and available in the art.

As used herein, “binds” is used interchangeably with “specificallybinds,” which means a polypeptide (e.g., a soluble receptor) or antibodywhich recognizes and hinds a polypeptide of the present disclosure, butthat does not substantially recognize and bind other molecules in asample, for example, a biological sample, which naturally includes apolypeptide of the present disclosure. In one example, an antibodyspecifically binds an activated STAT5 polypeptide does not hind anon-STAT5 polypeptide.

As used herein, “antibody” refers to an intact antibody orantigen-binding fragment of an antibody, including an intact antibody orantigen-binding fragment that has been modified or engineered, or thatis a human antibody.

In a particular embodiment, the antibody binds to and inhibits thefunction of any one or more of the factors that mediate T_(H)-GMfunction. For example, the antibody hinds to and inhibits the functionof IL-7, IL-7 receptor (IL-7R), IL-2, IL-2 receptor (IL-2R), STAT5 orjanus kinase 1/3 (JAK1/3), or a combination thereof. In other examples,the antibody hinds to and inhibits the function of GM-CSF (or itsreceptor), IL-3, PENK, or RANKL, or a combination thereof. In someembodiments, the antibody binds to and inhibits the function of a genelisted in Table 1. In some embodiments, the antibody hinds to andinhibits the protein or any functional fragment thereof. Methods ofdesigning, producing and using suitable antibodies are known andavailable to those of skill in the art. Examples of antibodies suitablefor use in the present disclosure include. e.g., daclizumab,basiliximab, mavrilimumab, MOR103, KB003, namilumab, and MOR Ab-022.

The terms “protein” and “polypeptide” are used interchangeably, and caninclude full-length polypeptide or functional fragments thereof (e.g.,degradation products, alternatively spliced isoforms of the polypeptide,enzymatic cleavage products of the polypeptide), the polypeptide houndto a substrate or ligand, or free (unbound) forms of the polypeptide.The term “functional fragment”, refers to a portion of a full-lengthprotein that retains some or all of the activity (e.g., biologicalactivity, such as the ability to bind a cognate ligand or receptor) ofthe full-length polypeptide.

In some embodiments, the modulating agent that inhibits T_(H)-GMfunction can be a particular biological protein (e.g., cytokines) thatinhibits, directly or indirectly, one or more of the factors thatmediate T_(H)-GM function. Such cytokines include, e.g., IL-12, IFN-γ,TGF-β, and IL-6.

In some embodiments, the modulating agent that inhibits T_(H)-GMfunction can be a small molecule that inhibits, directly or indirectly,one or more of the factors that mediate T_(H)-GM function. As usedherein a “small molecule” is an organic compound or such a compoundcomplexed with an inorganic compound (e.g., metal) that has biologicalactivity and is not a polymer. A small molecule generally has amolecular weight of less than about 3 kilodaltons. Examples of knownsmall molecules include CAS 285986-31-4 (Calbiochem), pimozide, andtofacitinib.

In other embodiments, the modulating agent enhances T_(H)-GM function indisorders such as, e.g., viral, fungal and bacterial infections, cancersand/or conditions associated with therewith. In one embodiment,modulating agents that enhance T_(H)-GM function include. e.g., CD28activator; IL-7 and/or IL-2 on naïve (precursor) CD4⁺ T cells; activatorof STAT5; or effectors of T_(H)-GM cells (e.g., GM-CSF. IL-3).

In another aspect, the present disclosure provides a method of treatinga STAT5-mediated inflammatory disorder in a patient in need thereof,comprising administering to the patient an effective amount of an agentthat modulates STAT5 function.

As used herein, “STAT5-mediated” inflammatory disorder refers to aninflammatory disorder that is caused by aberrant STAT5 function(aberrantly enhanced or inhibited), and which is responsive tomodulation of STAT5 function, as determined by clinical standards. Insome embodiments, the STAT5 is activated STAT5 (e.g., phospho-STAT5,Tyr694).

In some embodiments, the inflammatory disorder is an autoimmunedisorder. In certain embodiments, the inflammatory disorder can be anyinflammatory disorder mediated by STAT5 (e.g., activated STAT5), andincludes, but is not limited to rheumatoid arthritis, multiplesclerosis, ankylosing spondylitis. Crohn's disease, diabetes.Hashimoto's thyroiditis, hyperthyroidism, hypothyroidism, IrritableBowel Syndrome (IBS), lupus erythematosus, polymyalgia rheumatic,psoriasis, psoriatic arthritis. Raynaud's syndrome/phenomenon, reactivearthritis (Reiter syndrome), sarcoidosis, scleroderma. Sjögren'ssyndrome, ulcerative colitis, uveitis, or vasculitis.

In some embodiments, the term “patient” refers to a mammal, preferablyhuman, but can also include an animal in need of veterinary treatment,e.g., companion animals (e.g., dogs, cats, and the like), farm animals(e.g., cows, sheep, pigs, horses, and the like), and laboratory animals(e.g., rats, mice, guinea pigs, and the like).

In some embodiments, the agent inhibits STAT5 function (e.g., expressionand/or activity). Examples of agents that inhibit STAT5 (e.g., activatedSTAT5, Tyr694) are described herein.

In certain embodiments, the methods of the present disclosure furthercomprise administering to the patient a TNF-αc therapy. In certainembodiments, TNF-α therapy is administered in a patient determined tohave an inflammatory condition that is non-T_(H)-GM-mediated. Asdescribed herein, in certain embodiments, a TNF-α therapy isadministered if a quantified TNF-α level is increased by, e.g., at least40% relative to a reference level.

Examples of TNF-α therapy include those that are TNF-α-inhibitor based,and those that are non-TNF-α-inhibitor based. In particular,TNF-α-inhibitor based therapy includes etanercept, adalimuinab,infliximab, golimumab, and certolizumab pegol. Examples ofnon-TNF-α-inhibitor based therapy includes corticosteroid medications(e.g., prednisone), nonsteroidal anti-inflammatory drugs (e.g.,methotrexate), and JAK inhibitors (e.g., tofacitinib). Other examples ofnon-TNF-α-inhibitor based therapy include anakinra, abatacept, rituximaband tocilizumab.

The TNF-α therapy can be administered before, simultaneously with, oralter the administration of an effective amount of an agent thatmodulates T_(H)-GM function. Accordingly, an agent that modulatesT_(H)-GM function and the TNF-α therapy can be administered together ina single dose, or can be administered in separate doses, e.g., eithersimultaneously or sequentially, or both. The duration of time betweenthe administration of an agent that modulates T_(H)-GM function and aTNF-α therapy will depend on the nature of the therapeutic agent(s). Inaddition, an agent that modulates T_(H)-GM function and a TNF-α therapymay or may not be administered on similar dosing schedules. For example,the agent that modulates T_(H)-GM function and the TNF-α therapy mayhave different half-lives and/or act on different time-scales such thatthe agent that modulates T_(H)-GM function is administered with greaterfrequency than the TNF-α therapy, or vice-versa. The number of days inbetween administration of therapeutic agents can be appropriatelydetermined by persons of ordinary skill in the art according to thesafety and pharmacodynamics of each drug.

The identification of the T_(H)-GM cells as well as the identificationof genes differentially produced by T_(H)-GM cells relative to T_(H)1 orT_(H)17 enables the use of T_(H)-GM cells to identify novel therapeuticsfor modulating T_(H)-GM function, thereby enabling new therapeutics fortreating T_(H)-GM-mediated disorders (e.g., inflammatory disorders).Thus, in further aspects, the present disclosure provides a method ofscreening to identify a modulator of T_(H)-GM cell function, comprisingcontacting an isolated population of T_(H)-GM cells, or an isolatedpopulation of CD4+ precursor cells, with a candidate agent, andmeasuring a readout of T_(H)-GM function in the presence or absence ofthe candidate agent, wherein a change in the readout of T_(H)-GMfunction indicates that the candidate agent is a modulator of T_(H)-GMfunction.

As used herein, a candidate agent refers to an agent that may modulateT_(H)-GM function by modulating the function (e.g., expression and/oractivity) of a factor that mediates T_(H)-GM function. Such candidateagents include, e.g., an antibody, a peptide, a small molecule, anucleic acid (e.g., antisense, small interfering RNA molecule), or aprotein (e.g., cytokine), or a combination thereof. A candidate agentcan be designed to target any of the factors (at the protein and/ornucleic acid level) that mediate T_(H)-GM function, as described herein,including the genes listed in Table 1 (e.g., genes preferentiallyupregulated in T_(H)-GM cells, genes preferentiallyoverexpressed/underexpressed on the surface of T_(H)-GM cells).

As used herein, “readout” refers to any change (or lack of change) inT_(H)-GM function that can be measured or quantified. For example, acandidate agent can be assessed for its effect on, e.g., GM-CSFsecretion by T_(H)-GM cells, or its effect on the abundance of T_(H)-GMcells (through an effect on the commitment/development/proliferation ofT_(H)-GM cells), as described herein. Assays for determining suchreadouts are known and available in the art, and are exemplified herein.

In some embodiments, the change in the presence of the candidate agentis a reduction in the measurement of the readout, indicating aninhibition of T_(H)-GM function (e.g., decrease in GM-CSF or IL-3production, or decrease in the abundance of T_(H)-GM cells), therebyidentifying the candidate agent as an inhibitor of T_(H)-GM function.

In certain embodiments, the change in the presence of the candidateagent is an increase in the measurement of the readout, indicating anenhancement of T_(H)-GM function (e.g., increase in GM-CSF or IL-3production, or increase in the abundance of T_(H)-GM cells), therebyidentifying the candidate agent as an enhancer of T_(H)-GM function.

In some embodiments, the readout can be any one or more of the geneslisted in Tables 1 and 2 which are preferentially upregulated ordownregulated in T_(H)-GM cells. Thus, a candidate agent thatdownregulates a gene that is preferentially upregulated in a T_(H)-GMcell is a inhibitor of T_(H)-GM function. Similarly, a candidate agentthat upregulates a gene that is preferentially downregulated in aT_(H)-GM cell is an enhancer of T_(H)-GM function.

In certain aspects, the method of screening, if performed with precursorCD4+ cells, is performed under T_(H)-GM polarizing conditions, asdescribed herein. For example, the method can be performed in thepresence of IL-7/STAT5, TCR activation, CD28 co-stimulation, incombination with the blockade of IFN-gamma and IL-4.

Unless indicated otherwise, the definitions of terms described hereinapply to all aspects and embodiments of the present disclosure

The practice of the present disclosure includes use of conventionaltechniques of molecular biology such as recombinant DNA, microbiology,cell biology, biochemistry, nucleic acid chemistry, and immunology asdescribed for example in: Molecular Cloning: A Laboratory Manual, secondedition (Sambrook et al., 1989) and Molecular Cloning: A LaboratoryManual, third edition (Sambrook and Russel, 2001), jointly andindividually referred to herein as “Sambrook”); OligonucleotideSynthesis (M. J. Gait, ed., 1984); Animal Cell Culture (R. I. Freshney,ed., 1987); Handbook of Experimental Immunology (D. M. Weir & C. C.Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M.Miller & M. P. Calos, eds., 1987); Current Protocols in MolecularBiology (F. M. Ausubel et al., eds., 1987, including supplements through2001); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994);Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); TheImmunoassay Handbook (D. Wild. ed., Stockton Press NY, 1994);Bioconjugate Techniques (Greg T. Hermanson, ed., Academic Press, 1996);Methods of Immunological Analysis (R. Masseyeff, W. H. Albert, and N. A.Staines, eds., Weinheim: VCH Verlags gesellschaft mbH, 1993), Harlow andLane (1988) Antibodies, A Laboratory Manual, Cold Spring HarborPublications, New York. and Harlow and Lane (1999) Using Antibodies: ALaboratory Manual Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. jointly and individually referred to herein as “Harlow andLane”), Beaucage et al. eds., Current Protocols in Nucleic AcidChemistry (John Wiley & Sons, Inc., New York, 2000); and Aerawal, ed.,Protocols for Oligonucleotides and Analogs, Synthesis and Properties(Humana Press Inc., New Jersey, 1993).

EXEMPLIFICATION

Methods

Mice

Stat5^(f/f) mice were provided by L. Hennighausen (National Institute ofDiabetes and Digestive and Kidney Diseases). Stat3^(f/f) mice weregenerated as described². Cd4-Cre transgenic mice were purchased fromTaconic Farms. Rag2^(−/−) mice were obtained from Jean-Pierre Abastado(Singapore Immunology Network). All mice are on a C57BL/6 geneticbackground and housed under specific-pathogen-free conditions atNational University of Singapore. All experiments were performed withmice 6˜8 weeks old and approved by the Institutional Animal Care and UseCommittee of NUS.

Patients and Controls

Blood samples (n=47) and synovial fluid samples (n=3) were collectedfrom RA patients admitted to the Department of Rheumatology andImmunology, the Affiliated Drum Tower Hospital of Nanjing UniversityMedical School. All patients fulfilled the American College ofRheumatology criteria for the classification of RA. Age and gendermatched healthy controls (n=32) were obtained from Medical ExaminationCenter of the Affiliated Drum Tower Hospital. The study protocol wasapproved by the Ethics Committee of the Affiliate Drum Tower Hospital ofNanjing University Medical School.

In Vitro T Cell Differentiation

CD4⁺ T cells were obtained from spleens and lymph nodes by positiveselection and magnetic separation (Miltenyi Biotech), followed bypurification of naïve CD4⁺ T cell population(CD4⁺CD25⁻CD62L^(hi)CD44^(lo)) sorted with FACS Aria. Naïve CD4⁺ T cellswere stimulated with plate-hound anti-CD3 (3 μg/ml; BD Pharmingen) andanti-CD28 (1 μg/ml; BD Pharmingen) in presence of different combinationsof neutralizing antibodies and cytokines for 3˜4 days: for neutralconditions, no addition of any cytokine or neutralizing antibody; forT_(H) 1 conditions, IL-12 (10 ng/ml), and anti-IL-4 (10 μg/ml, BDPharmingen); for T_(H)17 conditions, hTGF-β (3 ng/ml), IL-6 (20 ng/ml),anti-IFN-γ (10 μg/ml, eBioscience), and anti-IL-4 (10 μg/ml); for analternative T_(H)17 conditions, IL-6 (20 ng/ml), IL-23 (10 ng/ml), IL-1(10 ng/ml), anti-IFN-γ (10 μg/ml), and anti-IL-4 (10 μg/ml). ForGM-CSF-expressing cell differentiation, naïve CD4⁺ T cells werestimulated with plate-bound anti-CD3 (2 μg/ml) and soluble anti-D28 (1μg/ml) with the addition of IL-7 and/or anti-IFN-γ (10 μg/ml) asindicated. All cytokines were obtained from R&D Systems. All cells werecultured in RPMI 1640 supplemented with 10% FBS, 100 units/mlpenicillin, 0.1 mg/ml streptomycin, 1 mM sodium pyruvate, 0.1 mMnonessential amino acid and 5 μM beta-mercaptoethanol. After 3˜4 dayspolarization, cells were washed and restimulated with phorbol12-myristate 13-acetate (PMA) and ionomycin in presence of Golgiplug for4-5 h, followed by fixation and intracellular staining with aCytofix/Cytoperm kit from BD Pharmingen. Foxp3 staining was done with akit from eBioscience. Cells were acquired on the LSR II (BD Biosciences)and analyzed with FlowJo software (Tree Star).

EAE Induction

EAE induction procedures were modified from previous report³. For activeEAE induction, mice were immunized in two sites on the hind flanks with300 μg MOG₃₅₋₅₅ in 100 μl CFA containing 5 mg/ml heat-killed M.tuberculosis strain H37Ra (Difco) at day 0 and day 7. Pertussis toxin(List Bio Lab) was administrated intraperitoneally at the dosage of 500ng per mouse at day 1 and day 8. For single MOG₃₅₋₅₅/CFA immunization,the similar procedure was performed at day 0 and day 1 only. In analternative active EAE induction, LPS (600 μg/ml in IFA, O111:B4 fromSigma) was used as adjuvant. For active EAE induction in Rag2^(−/−)mice, CD4⁺ T cells derived from Stat5^(f/f) or Cd4-Cre; Stat5^(f/f) micewere transferred, followed by MOG₃₅₋₅₅/CFA immunization as describedabove. Clinical symptoms were scored as follows: 0, no clinical sign; 1,loss of tail tone; 2, wobbly gait; 3, hind limb paralysis; 4, hind andfore limb paralysis; 5, death. IL-7Rα neutralizing antibody (SB/14, BDPharmingen) and isotype control was administrated intraperitoneally at200 μg per mouse every other day. For analysis of CNS-infiltratingcells, both spinal cord and brain were collected and minced fromperfused mice, and mononuclear cells were isolated by gradientcentrifuge with Percoll (GE Healthcare).

For passive EAE induction with Stat5^(+/+) or Stat5^(−/−) T cells,splenocytes and LNs were harvested 10-14 days post-immunization andpassed through a 70 μm cell strainer (BD Falcon). Cells were cultured invitro for 3 days with MOG₃₅₋₅₅ (20 μg/ml) in the presence of IL-23 (5ng/ml) and IL-1β (2 ng/ml). After harvesting, CD4⁺ T cells were purifiedby positive selection to a purity >90%. CD4⁺ T cells (2 million insterile PBS) were injected intraperitoneally into Rag2^(−/−) mice,followed by Pertussis toxin administration on the following day. Micewere observed daily for the signs of EAE as described above. For EAEinduction by transferring various T_(H) subsets, similar procedures wasperformed as described above. Different subsets skewing conditions wereas follows: Non-skewed, MOG₃₅₋₅₅ only; T_(H)1: MOG₃₅₋₅₅ plus IL-12 (10ng/ml) and anti-IL-4 (5 μg/ml); T_(H)17: MOG₃₅₋₅₅ plus TGF-β (3 ng/ml),IL-6 (10 ng/ml), anti-IFN-γ (5 μg/ml) and anti-IL-4 (5 μg/ml);GM-CSF-expressing T_(H): MOG₃₅₋₅₅ plus IL-7 (2 ng/ml) and anti-IFN-γ (5μg/ml). 6×10⁵ CD4⁺ T cells were transferred per recipient mouse.

Antigen-Induced Arthritis (AIA)

Briefly, mice were immunized subcutaneously in two sites on the hindflanks with 100 μg methylated bovine serum albumin (mBSA, Sigma) in 100μl complete Freund's adjuvant (CFA) containing 5 mg/ml heat-killed M.tuberculosis strain H37Ra (Difco) at day 0. Pertussis toxin (List BioLab) was administrated intraperitoneally at the dosage of 250 ng permouse at day 1. Arthritis was induced by intraarticular injection of 100μg mBSA (in 10 saline) into the hind right knee joint at day 7 afterimmunization. The hind left knee joint was injected with same volume ofsaline as control. Joint swelling was recorded by measuring thedifference between right and left knee joint diameters with a caliperover 7 days after arthritis induction. To assess the effect of GM-CSFadministration, MA was induced by intraarticular injection of mBSA aloneto the right knee joint or mBSA supplemented with 100 ng GM-CSF(ImmunoTools) to the left knee joint. To assess the effect of GM-CSFand/or TNF-α blockade, mice were administrated intraperitoneally withneutralizing antibodies (100 μg for each antibody per mouse) specificfor GM-CSF (MP1-22E9, BD Pharmingen) and/or TNF-α (MP6-XT3. BDPharmingen) at indicated times.

For AIA induction by adoptive transfer, splenocytes and inguinal LNcells were isolated from mBSA/CFA-immunized mice at day 7, and culturedin vitro with mBSA (10 μg/ml) in the presence of IL-7 (2 ng/ml) for 3days. After harvesting, CD4⁺ T cells were purified by positive selection(Miltenyi Biotec) to a purity >90%. Then CD4⁺ T cells (1 million insterile PBS) were transferred into WT naïve mice, followed byintraarticular injection of mBSA on the next day.

Collagen-Induced Arthritis (CIA)

CIA was induced in a similar procedure as AIA as described above, byimmunizing mice with chicken collagen II/CFA emulsion (purchased fromChondrex, Inc), followed with pertussis toxin injection. Mice weremonitored and scored for arthritis; 0, normal; 1, mild swelling of ankleor wrist, or apparent swelling limited to individual digits; 2, moderateswelling of entire paw; 3, severe swelling of entire paw with ankylosis.Scores for four limbs were summed for each mouse.

Histological Analysis

For paraffin-embedded tissues, spinal cords were fixed in 4% PFA. Kneejoints or paws were removed, fixed in 10% formalin and decalcified in 5%formic acid before dehydration and embedding. Sections (5 μm) werestained with hematoxylin and eosin (H&E) to assess immune cellinfiltration and inflammation, or with Safranin-O/Fast Green to assesscartilage depletion. For frozen tissues, spinal cords were embedded inOCT (Tissue-Tek) and snap frozen on dry ice. Sections (10 μm) were fixedin ice-cold acetone and stained with primary anti-CD4 (Biolegend) andanti-CD11b (eBioscience), followed by incubation withfluorescence-conjugated secondary antibodies (Invitrogen). For ALAexperiments, knee joint were fixed in 10% formalin for 5 days, followedby decalcification in 5% formic acid for 5 clays. Sections (10 μm) werestained with hematoxylin and eosin (H&E) to assess immune cellinfiltration and inflammation, or stained with Safranin-O/fast green toaccess cartilage destruction.

Cell Sorting and May Grünwald-Giemsa Staining

Monocytes/macrophages (Ly6C^(hi)Ly6G⁻) and neutrophils(Ly6C^(lo)Ly6G^(hi)) gated on CD45⁺CD11b⁺ were sorted with FACS Ariafrom spleens or synovial single cell suspensions. Sorted cells werecytospun onto glass slides and subsequently stained with May Grünwaldand Giemsa dye following a standard procedure.

Real-Time PCR

Total RNA was extracted from cells with RNeasy kit (Qiagen) according tothe manufacturer's instruction. Complementary DNA (cDNA) was synthesizedwith Superscript reverse transcriptase (Invitrogen). Gene expressionswere measured by 7500 real-time PCR system (Applied Biosystems) withSYBR qPCR kit (KAPA). Actinb, Gapdh or Rn18S was used as internalcontrol. The primer sequences are available upon request.

ELISA

TNF-α, IL-6, IL-1β, IFN-γ, GM-CSF and IL-2 levels were assayed byReady-SET-Go ELISA kit (eBioscience), and IL-17 level was measured byDuoSet ELISA kit (R&D Systems) according to the manufactures'instructions.

Chromatin Immunoprecipitation Assays

CD4⁺ T cells isolated from Stat5^(f/f) or Cd4-Cre; Stat5^(f/f) mice wereactivated with plate-bound anti-CD3 and anti-CD28 for 3 days. Cells werestimulated with IL-7 (20 ng/ml) or IL-2 (25 ng/ml) for 45 min. Crosslinkwas performed by addition of formaldehyde at final concentration of 1%for 10 min followed by quenching with Glycine. Cell lysates werefragmented by sonication and precleared with protein G Dynabeals, andsubsequently precipitated with anti-STAT5 antibody (Santa Cruz) ornormal rabbit IgG (Santa Cruz) overnight at 4° C. After washing andelution, crosslink reversal was clone by incubating at 65° C. for 8 hr.The eluted DNA was purified and analyzed by RT-PCR with primers specificto C42 promoter as described previously⁵.

Statistics

Statistical significance was determined by Student's t test usingGraphPad Prism 6.01. The p value <0.05 was considered significant. The pvalues of clinical scores were determined by two-way multiple-rangeanalysis of variance (ANOVA) for multiple comparisons. Unless otherwisespecified, data were presented as mean and the standard error of themean (mean±SEM).

Example 1. Stat5 Conditional Knockout Mice are Resistant to EAE

STAT5 negatively regulates T_(H)17 differentiation by restraining IL-17production (Laurence et al., 2007; Yang et al., 2011). However, thefunction of STAT5 in T_(H)17-mediated pathogenesis is not wellunderstood. To explore this question, EAE was induced in Cd4-Cre;Stat5^(f/f) (Stat5^(−/−)) mice, where Stat5 was specifically deleted inT cell compartment, and in littermate controls by immunizing the micewith MOG₃₅₋₅₅/CFA at day 0 and day 7. Development of paralysis wasassessed by daily assignment of clinical scores. Surprisingly,diminished occurrence and severity of clinical disease in Stat5^(−/−)mice was observed (FIGS. 1A and 1B), a result that was opposite toexpectations based on an antagonistic role for STAT5 in T_(H)17generation. Similar results were observed when a single MOG₃₅₋₅₅/CFAimmunization was performed or replaced CFA with LPS as the adjuvant toinduce EAE (FIGS. 1C and 1D). Consistent with reduced EAE disease inStat5^(−/−) mice, a remarkable reduction of immune cell infiltration inthe spinal cord of Stat5^(−/−) mice was observed (FIG. 2A). Furthermore,the infiltration of various immune cell populations, including CD4⁺,CD8⁺. B220⁺ and CD11b⁺ cells was reduced in Stat5^(−/−) mice (FIGS. 2B-Dand data not shown). However, the frequencies of IL-17⁺ and IFN-γ⁺ cellsamong CD4⁺ T cells in the CNS were comparable between Stat5^(+/+) andStat5^(−/−) mice (FIG. 3A), suggesting the resistance to EAE inStat5^(−/−) mice is independent of T_(H)1 and T_(H)17 cell development.Nevertheless, decreased CD4⁺CD25⁺ T_(reg) population in the CNS ofStat5^(−/−) mice was detected (FIG. 3B), indicating the resistance toEAE in Stat5^(−/−) mice was unlikely due to altered T_(reg), cells.

Example 2. Resistance to EAE in Stat5-Mutant Mice is Due to an IntrinsicDefect of Antigen Specific CD4⁺ T Cells Independent of T_(H)1 andT_(H)17 Generation

Stat5 deletion (Cd4-cre; Stat5^(f/−)) mice was reported to developperipheral lymphopenia, with a reduction of both CD4⁺ and CD8⁺ T cells(Yao et al., 2006). However, another study showed that Stat5 deletion(Cd4-cre; Stat5^(f/f)) did not affect the proportion of peripheral CD4⁺T cells (Burchill et al., 2007). In the experimental setting, a changein the absolute number of peripheral CD4⁺ T cells was not detected byStat5 deletion during EAE development (FIGS. 4A and 4B), suggesting theresistance to EAE in Stat5^(−/−) mice was not caused by peripherallymphopenia. Furthermore, increased frequencies of IL-17⁺ and IFN-γ⁺cells were detected among CD4⁺ T cells in spleens of Stat5^(−/−) mice(FIG. 4C), which further support the idea that the resistance to EAE inStat5^(−/−) mice is likely independent of T_(H)1 and T_(H)17 generation.To validate the function of STAT5 in T_(H)1 and T_(H)17 generation, thein vitro differentiation was performed by activating naïve CD4⁺ T cellsunder T_(H)1- and T_(H)17-polarizing conditions. In agreement withprevious reports, that STAT5 mediated the suppressive effect of IL-2 onT_(H)17 differentiation (data not shown). Interestingly, IL-7, whichalso signals through STAT5, was not observed to have a demonstrableeffect on T_(H)17 differentiation (data not shown). Nevertheless, aslight decrease of IFN-γ⁺ cells under T_(H)1-polarizing condition wasobserved when STAT5 was deleted (data not shown).

To confirm if the resistance of EAE in Stat5^(+/+) mice is mediated byCD4⁺ T cells, Rag2^(−/−) mice were reconstituted with Stat5^(+/+) orStat5^(−/−) CD4⁺ T cells followed by EAE induction. We found thatRag1^(−/−) mice that received Stat5^(−/−) CD4⁺ T cells were resistant tothe disease compared with mice receiving wild-type cells (data notshown), demonstrating that Stat5^(−/−) CD4⁺ T cells were impaired intheir ability to promote EAE development.

Next, whether the lack of encephalitogenicity was caused by defects inmigration of Stat5^(−/−) CD4⁺ T cells to the CNS was examined. It hasbeen shown that the chemokine receptor CCR6 is essential for T_(H)17cell entry into the CNS through the choroid plexus (Reboldi et al.,2009). Thus. CCR6 expression in both Stat5^(−/−) and Stat5^(+/+) CD4⁺ Tcells was examined. Increased CD4⁺CCR6⁺ cells in spleens of Stat5^(−/−)mice compared with Stat5^(+/+) controls (FIG. 5A) was observed. CXCR3and CD69 expression was also examined, which showed increased expressionof both molecules in CD4⁺ T cells in the absence of STAT5 (FIG. 5A).These results indicate that Stat5^(−/−) CD4⁺ T cells can infiltrate CNS.Furthermore, comparable number of CD4⁺ T cells present in the CNS ofStat5^(+/+) and Stat5^(−/−) mice during EAE induction was observed (atday 7 and day 9) (FIG. 5B). However, CD4⁺ T cells in CNS of Stat5^(−/−)mice dropped dramatically during disease onset (Day 21) (FIGS. 5C and5D). Together, these results demonstrate that Stat5^(−/−) CD4⁺ T cellscan infiltrate CNS, but fail to induce effective inflammation in the CNSin EAE.

To further exclude the possibility that the resistance ofStat5-deficient mice to EAE was caused by any potential defect in thesurvival of autoreactive CD4⁺ T in the CNS, increased numbers ofStat5^(−/−) CD4⁺ T cells than wild-type cells were transferred intoRag1^(−/−) mice respectively to make sure comparable numbers ofautoreactive CD4⁺ T cells were present in the CNS during EAEdevelopment. As shown in FIGS. 6A and 6B, despite similar numbers ofCD4⁺ T cells in the CNS between two groups of mice, reduced diseaseseverity was nevertheless observed in mice receiving Stat5-deficientCD4⁺ T cells. Additionally, certain numbers of Stat5-deficient micecontaining similar numbers of CD4⁺ T cells in the CNS as wild-type miceat peak of EAE disease were observed, yet, they were relativelyresistant to EAE compared with those wild-type mice (FIG. 6C), furthersuggesting that the resistance to EAE disease in Stat5-deficient micewas unlikely due to impaired CD4⁺ T cell survival in the CNS.

To further develop a causal link between these observations and theintrinsic impairment of Stat5^(−/−) CD4⁺ T cells, MOG₃₅₋₅₅-specificStat5^(+/+) and Stat5^(−/−) CD4⁺ T cells were transferred intoRag2^(−/−) mice separately without further immunization to test if thesecells were able to mediate EAE development. As shown in FIGS. 7A and 7B,mice receiving Stat5^(+/+) CD4⁺ T cells spontaneously developed EAEdisease 1 week after transfer. In contrast, mice receiving Stat5^(−/−)CD4⁺ T cells had significantly reduced disease severity and incidence.The frequencies of IL-17⁺ and IFN-γ⁺ cells among CD4⁺ T cells in the CNSof Rag2^(−/−) mice were comparable between two groups (FIG. 7C), furthersuggesting that the intrinsic defect in encephalitogenicity ofStat5^(−/−) CD4⁺ T cells is independent of T_(H)1 and T_(H)17. Toexclude the possible role of CD8⁺ T cells in the resistance to EAEobserved in Stat5⁻ mice, Rag2^(−/−) mice were reconstituted withMOG₃₅₋₅₅-specific Stat5⁺⁴ or Stat5^(−/−) CD4⁺ T cells together withequal numbers of Stat5^(+/+) CD8⁺ T cells. The transfer of Stat5^(−/−)CD4⁺ together with Stat5^(+/+) CD8⁺ T cells still failed to induce EAE(data not shown). Together, these data demonstrate that Stat5^(+/+) CD4⁺T cells have intrinsic defect in encephalitogenicity. The relevantteachings of all patents, published applications and references citedherein are incorporated by reference in their entirety.

Example 3. Diminished Expression of GM-CSF in Stat5^(−/−) CD4⁺ T Cells

To test whether GM-CSF production was impaired by Stat5 deletion, itsexpression was examined in MOG₃₅₋₅₃-specific Stat5^(+/+) and Stat5^(+/+)CD4⁺ T cells. Splenocytes derived from MOG₃₅₋₅₅/CFA-immunizedStat5^(+/+) and Stat5^(−/−) mice were challenged with variousconcentrations of MOG₃₅₋₅₅ for 24 h, to examine the secretion of GM-CSF.GM-CSF production was observed to increase in a MOG₃₅₋₅₅ dose-dependentmanner in Stat5^(+/+) cells (FIG. 8A). In contrast, GM-CSF productionwas severely diminished in Stat5^(+/−) cells in all conditions. Tofurther validate this, splenocytes derived from mice were stimulatedduring the development of EAE with PMA/Ionomycin in the presence ofGolgiPlug for GM-CSF and IL-17 intracellular staining. Although IL-17expression was enhanced in Stat5^(−/−) cells, a significantly reducedproportion of GM-CSF⁺IL-17″ and GM-CSF⁺IL-17⁺ cells was observed amongCD4⁺CD44^(hi) cells in the absence of STAT5 (FIG. 8B). Moreover, thefrequency of MOG_(35,55)-specific GM-CSF⁺ T cells was also significantlyreduced in spleens of Stat5^(−/−) mice (FIG. 8C). Together, theseresults indicate that STAT5 is required for GM-CSF expression inautoreactive CD4⁺ T cells. However, STAT5, an important transcriptionfactor in T_(H)17 differentiation, was required for GM-CSF expression(FIG. 8D).

Next. GM-CSF induction in the CNS during EAE development was examined.Although IL-17 and IFN-γ production by CNS-infiltrating CD4⁺ T cells wasnot impaired by Stat5 deficiency, a diminished frequency of CD4⁺GM-CSF⁺cells in the CNS of Stat5^(−/−) mice was detected compared with controlmice (FIG. 9A). Further analysis showed a reduced GM-CSF⁺ percentageamong CD4⁺IL-17; cells and among CD4⁺IFN-γ⁺ cells (FIG. 9A). Similarly,Rag2^(−/−) mice transferred with MOG₃₅₋₅₅-specific Stat5^(−/−) CD4⁺ Tcells also showed a reduced frequency of CD4⁺GM-CSF⁺ T cells in the CNScompared with control mice (FIG. 9B). GM-CSF mRNA expression in the CNSof Stat5^(−/−) mice was markedly lower than that of Stat5^(+/+) mice atday 8 after EAE induction (FIG. 9C), when comparable CD4⁺ T cellinfiltration was detected in Stat5^(−/−) and Stat5^(+/+) mice (FIGS. 5Cand 5D). Meanwhile, no significant difference of IL-17 and IFN-γexpression was detected between Stat5^(−/−) and Stat5^(+/+) mice (FIG.9C). The impaired cytokine induction (IL-17 and IFN-γ) in the CNS ofStat5^(−/−) mice at later stage (day 14, FIG. 9C) could be explained bythe inability of Stat5^(−/−) CD4⁺ T to sustain neuroinflammation (FIGS.5C and 5D). Interestingly, GM-CSF induction in the CNS preceded IL-23induction (FIG. 9C), suggesting IL-23 might not be required for GM-CSFexpression in the induction phase of EAE. In summary, the resultssuggest that GM-CSF expression in autoreactive CD4⁺ T cells is regulatedby STAT5 and the impaired GM-CSF production in the absence of STAT5caused the resistance of the mice to EAE.

Example 4. IL-7-STAT5 Signaling Induces GM-CSF Expression inAutoreactive CD4⁺ T Cells and Contributes to Neuroinflammation

Next, the mechanism by which STAT5 regulates GM-CSF expression wasinvestigated. As the present disclosure indicates, neither IL-23 norIL-1β seemed to be potent STAT5 stimulators (FIG. 10A). Furthermore,IL-1R1 expression was not changed, whereas IL-23Rα expression wasincreased in Stat5^(−/−) CD4⁺ T cells (FIG. 10B). These data suggestthat the ability of STAT5 to induce GM-CSF expression is likelyindependent of IL-23 and IL-1β signaling. In contrast, both IL-2 andIL-7 potently activated STAT5 by inducing tyrosine phosphorylation (FIG.10A). Therefore, the effect of these two cytokines on GM-CSF inductionin autoreactive T cells was further examined. Splenocytes derived fromMOG₃₅₋₅₅-immunized wild-type mice were challenged with MOG₃₅₋₅₅ alone orplus IL-2. GM-CSF and IL-17 production by CD4⁺ T cells were analyzed byintracellular cytokine staining. As shown in FIG. 10C, IL-2 showedmodest effects on the frequency of GM-CSF⁺ T cells. In contrast, IL-7significantly promoted GM-CSF expression in both IL-1T and IL-17⁺CD4⁺CD44^(hi) T cells (FIG. 11A). Furthermore, IL-7 carried out thisfunction in a STAT5-dependent manner, as Stat5 deletion abrogated itseffect on GM-CSF expression as assessed by intracellular cytokinestaining and ELISA (FIG. 11A, lower panels, and FIG. 11B).

IL-7Rα is expressed in both CD62L^(hi)CD44^(lo)T cells andCD62^(lo)CD44^(hi) T cells, suggesting IL-7 may directly act on CD4⁺ Tcells to regulate GM-CSF expression. Thus, CD62L^(hi)CD44^(lo) andCD62L^(lo)CD44^(hi) T cells were sorted from Stat5^(−/−) mice andlittermate controls during EAE development, and then activated cells inthe presence or absence of IL-7. As shown in FIG. 11C,CD62L^(lo)CD44^(hi) T cells potently expressed GM-CSF, whileCD62L^(hi)CD44^(lo)) T cells expressed 30-fold lower GM-CSF amounts.STAT5 deletion resulted in reduced basal GM-CSF production inCD62L^(lo)CD44^(hi) T cells. As expected, IL-7 promoted GM-CSFexpression in both subsets of CD4⁺ T cells in a STAT5-dependent manner(FIG. 11C).

To examine the contribution of IL-7-induced GM-CSF expression inautoreactive CD4⁺ T cells to EAE development, mice were treated withIL-7Rα-specific antibody (clone SB/14) during EAE development. Thetreatment resulted in a significant reduction of disease severity, whichwas accompanied with reduced CNS inflammation (FIGS. 12A and 12B). Inagreement with previous report (Lee et al., 2012), this neutralizingantibody did not have T cell depleting activity (FIG. 12C). Notably, theblocking of IL-7 signaling resulted in decreased GM-CSF expression inCNS-infiltrating CD4⁺ T cells (FIGS. 120-12F). In summary, the presentfindings indicate that IL-7 induces STAT5-dependent GM-CSF expression inautoreactive CD4⁺ T cells, which contributes to the development ofneuroinflammation.

Example 5. GM-CSF-Expressing T_(H) Cells are Distinct from T_(H)17 andT_(H)1

Since both T_(H)17 and T_(H)1 can produce GM-CSF, it was determined ifthe IL-7-stimulated phenotype was related to either of these subsets. Tofurther understand the characteristics of GM-CSF-expressing CD4⁺ cells,naïve CD4⁺ T cells were stimulated with plate-bound anti-CD3 and solubleanti-CD28 under T_(H)1- or T_(H)17-polarizing conditions. It wasobserved that anti-CD3 together with anti-CD28 induced the expression ofGM-CSF (FIG. 14A). However, while T_(H)1 differentiation conditionspromoted IFN-γ expression and T_(H)17 conditions promoted IL-17expression as expected, both T_(H)1 and T_(H)17 differentiationconditions greatly suppressed the production of GM-CSF (FIGS. 13A and13B). Conversely, IL-12 and IFN-γ neutralization promotedGM-CSF-expressing cell generation (FIG. 13A), consistent with a previousreport (Codarri et al., 2011). IL-23 and IL-1β did not increaseGM-CSF-expressing cell differentiation from naïve CD4⁺ T cells (FIG.13A), which was consistent with the finding that naïve CD4⁺ T cells didnot express their receptors. TGF-β inhibits GM-CSF expression (El-Behiet al., 2011). IL-6, an essential cytokine for T_(H)17 differentiation,had a profound inhibitory effect on GM-CSF expression (FIG. 14B),indicating STAT3 could be a negative regulator. To address this, naïveCD4⁺ T cells were purified from Stat3^(−/−) mice for celldifferentiation. Strikingly, in the absence of STAT3, cells polarizedunder T_(H)17 condition expressed GM-CSF (FIG. 14C). Interestingly, evenwithout exogenous IL-6, STAT3 still had a moderate suppressive effect onGM-CSF-expressing cell differentiation (FIG. 14C). In addition, RORγtand T-bet have been reported unnecessary for GM-CSF expression in CD4⁺ Tcells (El-Behi et al., 2011). Thus, the present datasupport a modelwherein GM-CSF-expressing CD4⁺ T cells develop via a lineage distinctfrom T_(H)17 and T_(H)1.

Example 6. IL-7-STAT5 Programs GM-CSF-Expressing T_(H) CellDifferentiation

The present findings disclosed herein (including .g., diminished GM-CSFexpression in Stat5^(−/−) CD4⁺ T cells in vivo, IL-7/STAT5-mediatedinduction of GM-CSF expression in naïve CD4⁺ T cells, and the distinctfeatures of GM-CSF-expressing T_(H) cells versus T_(H)1 and T_(H)17cells) indicates a distinct T_(H) cell subset that is regulated byIL-7-STAT5 signaling. This finding was further explored by examiningGM-CSF-expressing T_(H) cell differentiation in vitro by activatingnaïve CD4⁺ T cells with anti-CD3 and anti-CD28 in the presence ofdifferent concentrations of IL-7. As shown in FIGS. 15A and 15B, IL-7strongly promoted the generation of GM-CSF-expressing cells and GM-CSFsecretion. Moreover, the generation of GM-CSF-expressing T_(H) by IL-7was mediated by STAT5. Without STAT5, IL-7 was unable to promote thegeneration of GM-CSF-expressing cells (FIGS. 15C and 15D). Furtherinvestigation showed that IL-7-induced STAT5 activation directly houndpromoter regions of the Csf2 gene (FIGS. 15E and 15F).

Small proportions of IFN-γ-expressing cells were generated duringGM-CSF-expressing T_(H) differentiation (FIG. 1.5A). Thus, the effect ofblocking IFN-γ on GM-CSF-expressing cell generation was tested, whichshowed that the combination of IL-7 and IFN-γ neutralization induced thehighest frequency of GM-CSF⁺ cells, where few IL-17⁺ or IFN-γ⁺ cellswere detected (FIG. 16A). Moreover, the expression of subset definingtranscriptional factors in GM-CSF-expressing T_(H) was examined andobserved that the expression of RORγt or T-bet in GM-CSF-expressingT_(H) was significantly lower than those in T_(H17) or T_(H)1 cells,respectively (FIG. 16B), confirming that the GM-CSF-expressing T_(H)cells are distinct from T_(H)1 and T_(H)17 cells. Together, these datasuggest that IL-7-STAT5 signaling direct the differentiation of a novelGM-CSF-expressing helper T cell subset, termed T_(H)-GM.

Next, it was determined whether IL-2 signaling could influence T_(H)-GMdifferentiation from naïve CD4⁺ T cells. The addition of IL-2 orantibody against IL-2 only had modest effect on the frequency of GM-CSF⁺cells (FIG. 14D), indicating a minimal effect of IL-2 on T_(H)-GMdifferentiation. Unlike IL-7Rα, IL-2 high-affinity receptor IL-2Ra wasnot expressed in naïve CD4⁺ T cells, but its expression was graduallyinduced by TCR activation (FIGS. 17A-17C). Thus, the minimal effect ofIL-2 at least in part is due to the unresponsiveness of naïve CD4⁺ Tcells to IL-2 stimulation. In support of this view, IL-7, but not IL-2,induced STAT5 activation and upregulated GM-CSF mRNA expression in naïveCD4⁺ T cells (FIGS. 17D and 17E). To further confirm this idea,activated CD4⁺ T cells were stimulated with IL-2 or IL-7, which showedthat both cytokines induced STAT5 activation, Csf2 promoter binding andGM-CSF mRNA upregulation (FIGS. 18A-18C). Notably, IL-2 induced aprolonged STAT5 activation compared with IL-7 (FIG. 18A).

Example 7. Distinct Gene Expression Profile of T_(H)-GM

To demonstrate T_(H)-GM as distinct from known T cell subsets (e.g.,T_(H)1 and T_(H)17), a whole transcriptome analysis was performed bymicroarray to validate its specificity compared with known T cellsubsets, in particular T_(H)17 cells. Naïve CD4⁺ T cells weredifferentiated into T_(H)1, T_(H)17 and T_(H)-GM. Microarray analysiswas performed to examine their gene expression profiles. Wholetranscriptome clustering indicates T_(H)-GM cells as representing anovel subset distinct from T_(H)1 or T_(H)17 cells. T celllineage-specific gene expression is shown in Table 1. A list of 202genes preferentially expressed in T_(H)1 cells were identified, comparedwith naïve, T_(H)17 or T_(H)-GM cells (fold change >1.7), among whichIFN-γ and T-bet are on the top of the list (Table 1). Similarly,T_(H)17-feature genes, such as IL-17, IL-17F, RORγt and RORα, wereidentified in the list including 411 genes specific to T_(H)17 cells(Table 1). The T_(H)-GM cell-specific gene list (“Genes preferentiallyupregulated in T_(H)-GM” the T_(H)-GM signature genes) contains 210genes including the gene encoding GM-CSF as the top gene in the list(Table 1). A set of surface molecules which were selectively expressedat high level in T_(H)-GM subset, and another set of surface moleculeswhich were selectively expressed at low level in T_(H)-GM subsetcompared with other subsets were identified (FIG. 19 and Table 1). Thesemolecules (also T_(H)-GM signature genes) can be used for furthercharacterization by surface markers to identify the T_(H)-GM subset of Tcells. Several other genes of interest were also identified, includinggenes encoding cytokines and transcriptional factors, in particularIL-3. Various helper T cells were differentiated in vitro and confirmedthat T_(H)-GM cells are potent IL-3 producers as compared with T_(H)1and T_(H)17 cells (FIGS. 20A, 20C and 20D). In addition, several othercytokines, including EBI-3, PENL and RANKL were found preferentiallyexpressed in T_(H)-GM cells (FIG. 20B), indicating diverse biologicalfunctions of T_(H)-GM cells.

Example 8. T_(H)-GM Cells are the Primary Pathogenic Population

To test the hypothesis that GM-CSF-expressing T_(H) subset (T_(H)-GM)was the primary encephalitogenic effector cells, adoptive transfer ofdifferent subsets of MOG₃₅₋₅₅-specific CD4⁺ T cells was performed intoRag2^(−/−) mice for EAE induction. As shown in FIG. 21,GM-CSF-expressing T_(H) cells were preferentially able to induce EAEcompared with T_(H)17 and T_(H)1 subsets.

Example 9. The Suppression of STAT5 Activity by Chemical InhibitorAttenuates GM-CSF Expression by T_(H)-GM and Ameliorates EAE

The effect of disrupting STAT5 activation by chemical inhibitor wasexamined to explore possible methods of treating autoimmuneneuroinflammation. The phosphorylation on the key tyrosine residue inSH2 domain is crucial for STAT5 activation and function. A commercialSTAT5 inhibitor (CAS 285986-31-4, Calbiochem) has been reported toselectively disrupt tyrosine phosphorylation and DNA binding of STAT5(Muller et al., 2008). First, the inhibitory effect of this inhibitor onSTAT5 activation upon IL-7 stimulation in CD4+ T cells was tested. At aconcentration of 50 μM, the inhibitor had about 50% inhibitory effect,which was further enhanced with the increase of concentration (FIG.22A). STAT5 inhibitor had low affinity and thus required a highconcentration to fully block STAT5 activation, whereas JAK3 inhibitorshowed potent inhibitory effect even at low concentration (FIG. 22B).The specificity of STAT5 inhibitor was next tested by examining itseffect on the activation of STAT3 and STAT1. As shown in FIGS. 22C and22D, this STAT5 inhibitor at relatively lower concentration (50 or 100μM) showed minimal inhibitory effect on both STAT3 and STAT1 activation.

The effect of STAT5 inhibition on T_(H)-GM differentiation was examined.As shown, STAT5 inhibitor suppressed T_(H)-GM differentiation in adosage-dependent manner (FIG. 22E). Reduced T_(H)1 differentiation uponSTAT5 inhibitor treatment was observed (data not shown), but T_(H)17differentiation was not suppressed by STAT5 inhibitor (data not shown).

To explore the therapeutic effect of targeting STAT5 activation in EAEdisease, the commercial STAT5 inhibitor was administered to wild-typemice intraperitoneally every other clay after disease onset. Developmentof paralysis was assessed by daily assignment of clinical scores. STAT5inhibition ameliorated EAE severity, associated with reduced immune cellinfiltration in the CNS (FIGS. 23A and 23B). In contrast, although JAK3inhibitor can potently block STAT5 activation (FIG. 22B), it showeddetrimental effect on EAE (FIG. 23B). Of note, STAT5 inhibitor resultedin reduced GM-CSF production in CNS-infiltrating CD4⁺ T cells (FIGS. 23Cand 23D). This study indicates that targeting STAT5 by chemicalinhibitor is useful in therapeutic intervention in MS.

Example 10. GM-CSF-Producing T_(H) Cells are Associated with Human RA

Plasma concentrations of GM-CSF and TNF-α in peripheral blood of RApatients were examined in comparison with gender/age-matched healthycontrol (HC), and found that both cytokines were elevated in RA (FIG.24A). Ex vivo frequencies of IFN-γ-, IL-17- or GM-CSF-producing T_(H)cells were quantified in RA and HC. High frequencies of IFN-γ- and/orGM-CSF-producing T_(H) cells were detected in all samples, but observedlow frequency (<1%) of IL-17-producing T_(H) cells (FIG. 24B).GM-CSF-single-producing (GM-CSF⁺IFN-γ⁻) T_(H) cells represented asubstantial population in both RA and HC (FIG. 24B). More importantly,the frequency of this population in peripheral blood of RA wassignificantly higher than that of HC (FIG. 24C). In contrast, neitherGM-CSF/IFN-γ-double-producing nor IFN-γ-single-producing T_(H) cellsshowed any significant difference in their frequencies between RA and HC(FIG. 24C). Therefore, the frequency of GM-CSF-single-producing T_(H)cells in peripheral blood is selectively elevated in RA, suggesting afunctional association of T_(H)-cell-secreted GM-CSF with RA. Moreover,a significant correlation between plasma GM-CSF concentration andGM-CSF-single-producing T_(H) cell frequency was observed in RA (FIG.24D).

To further evaluate the association of GM-CSF-producing T_(H) cells withRA, mononuclear cells were isolated from synovial fluid of RA patientsand analyzed the abundance of these cells. A marked elevation ofGM-CSF-producing T_(H) cell frequency was observed in synovial fluidcompared with peripheral blood, but most of these cells co-expressedIFN-γ (FIG. 24E). Similarly, both T_(H)1 and T_(H)17 frequencies werealso increased in synovial fluid, with T_(H)17 remaining to be a minorpopulation compared with T_(H)1 (FIG. 24E).

Example 11. GM-CSF Mediates Experimental Arthritis in aTNF-α-Independent Manner

The elevation of GM-CSF and TNF-α level in plasma of RA in comparison toHC may suggest a therapeutic approach by targeting these two cytokines.The efficacy of blocking both GM-CSF and TNF-α was tested in treatingarthritic mice in antigen-induced arthritis (AIA) model, which is aT-cell driven RA model and is easily inducible in C57BL/6 strain with arapid and synchronized disease onset, facilitating the exploration of RApathogenesis. Either GM-CSF or TNF-α individual blockade attenuated MAdevelopment (FIG. 25A). Interestingly, the combination of GM-CSF- andTNF-α-specific neutralizing antibodies showed better efficacy incontrolling arthritis development than individual treatments (FIG. 25A).That is, targeting GM-CSF may have beneficial efficacy in treatingarthritis in a way independent of TNF-α activity. To further study thedistinguishable effects of GM-CSF and TNF-α in mediating arthritisdevelopment, a mouse strain (Cd4-Cre; Stat5^(f/f), or Stat5^(−/−) inshort) with conditional Stat5 deletion was used in T cells for AIAinduction. These conditional Stat5-knockout mice resisted arthritisdevelopment, as exemplified by milder joint selling, fewer immune cellinfiltration in synovia, and reduced joint destruction (FIGS. 25B-25D),even though they had markedly increased level of serum TNF-α as well asIFN-γ (FIG. 25E). In contrast, serum level of GM-CSF was significantlyreduced in knockout mice (FIG. 25E), which was likely the causal factorof the resistance to arthritis development as further supported byresults described below. Consistent results were also observed incollagen-induced arthritis (CIA) model (FIGS. 26A-26D). Together, thesefindings suggest that GM-CSF is an important pathogenic mediator in RAand also indicate the promise of developing anti-GM-CSF drugs to treatRA patients who are anti-TNFα drugs unresponsive, markingGM-CSF-producing T_(H) cells as a new biomarker for RA diagnosis.

Example 12. STAT5-Regulated GM-CSF Secretion by Autoreactive T_(H) CellsMediates Synovial Inflammation

On the basis of association of GM-CSF with RA, the cellular producers ofGM-CSF and the regulatory mechanism underlying GM-CSF expression inarthritic mice were examined. Splenocytes were collected from wild-typeAIA mice and separated cells into three fractions: splenocytes,splenocytes depleted of CD4⁺ T cells and CD4⁺ T cells; and stimulatedeach fraction at same cell numbers under various conditions. Splenocytesproduced low but detectable level of GM-CSF without stimulation, whichwas markedly increased by PMA/Ionomycin or mBSA antigen stimulation(FIG. 28A). Under all conditions, splenocytes depleted of CD4⁺ T cellsalmost completely abrogated GM-CSF production (FIG. 28A). In contrast,CD4⁺ T cells produced dramatically elevated GM-CSF in comparison tosplenocytes under all conditions (FIG. 28A). These results stronglysupport that CD4⁺ T cells are predominant producers of GM-CSF at leastin spleens of arthritic mice, which is somehow consistent the observedcorrelation of plasma GM-CSF concentration with GM-CSF-single-producingT_(H) cell frequency in RA (FIG. 24D). Thus, the functional significanceof T_(H)-cell-secreted GM-CSF was examined in arthritis development.Given T-cell-specific Csf2-knockout mice is not available and STAT5 is akey regulator of GM-CSF expression in T_(H) cells, conditionalStat5-knockout mice was used, which showed decreased GM-CSF level andresistance to arthritis development as described above.

Consistent with a previous study (Burchill et al., 2007), similarfrequencies of CD4⁺ T cells were observed in peripheral lymphoid tissuesas well as in inflamed synovial tissues of STAT5-deficient mice comparedwith wild-type mice at day 7 after AIA induction (FIGS. 27A-27D),suggesting loss of STAT5 seems to not impair CD4⁺ T-cell generation inperiphery and infiltration in synovial tissues. To determine therequirement of STAT5 for arthritogenic potential of CD4⁺ T cells, exvivo-expanded antigen-reactive CD4⁺ T cells, derived from Stat5^(+/+)and Stat5^(−/−) ALA mice, were transferred into wild-type naïve miceseparately, followed by intra-articular injection of mBSA. Micereceiving Stat5^(+/+) CD4⁺ T cells displayed an abundant immune cellinfiltration in synovial tissues at day 7 after AIA induction (FIG.27E). In contrast, mice receiving Stat5^(−/−) CD4⁺ T cells had markedreduction of synovial infiltrates (FIG. 27E). Therefore, STAT5-deficientCD4⁺ T cells are defective in arthritogenic potential.

Multiple lines of evidence support a central role of T cells in RA.However, the pathogenic mechanism of T cells remains insufficientlyunderstood. Although T_(H)1 is a predominant population among synovialinfiltrating CD4⁺ T cells in human RA (Berner et al., 2000; Yamada etal., 2008), defective IFN-γ signaling results in increased diseasesusceptibility in animal models of arthritis (Guedez et al., 2001;Irmler et al., 2007; Manoury-Schwartz et al., 1997; Vermeire et al.,1997). In contrast, T_(H)17 cells are proven crucial in animal models ofarthritis (Pernis, 2009), but predominance of T_(H)17 cells is limitedin both peripheral blood and synovial compartment of human RA (Yamada etal., 2008) and (FIGS. 1B and 1E). As demonstrated herein,STAT5-regulated GM-CSF-producing T_(H) cells potentiate arthritispathogenesis.

To validate the regulatory role of STAT5 in GM-CSF production,splenocytes derived from AIA mice were stimulated with PMA/Ionomycinplus Golgiplug ex viva, followed by intracellular cytokine staining andflow cytometry. As expected, the frequency of GM-CSF-single-producingcells among CD4⁺CD44^(hi) population was significantly decreased inStat5^(−/−) mice (FIG. 28 34B). Notably, no significant differences wereobserved in frequencies of IL-17-single-producing (T_(H)17) orIFN-γ-single-producing (T_(H)1) cells between two groups (FIG. 28B).Further study by combining mBSA restimulation and intracellular cytokinestaining showed that the frequency of mBSA-specific GM-CSF-producingeffector T cells was much lower in spleens of Stat5^(−/−) mice thanthose in controls (FIG. 29A). In addition, AIA mice-derived splenocytesand inguinal lymph nodes (LNs) were restimulated with mBSA ex viva tomeasure cytokine concentrations in culture supernatants and found asignificant reduction of GM-CSF with deletion of STAT5, but comparablelevels of both IL-17 and IFN-γ between two groups (FIGS. 29B and 29C).Together, the results indicate that loss of STAT5 may specificallysuppress GM-CSF-producing effector Th cells but not T_(H)17 or T_(H)1cells in experimental arthritis.

To investigate the involvement of GM-CSF-producing T_(H) cells and theirregulation by STAT5 in synovial inflammation, synovial tissues weredissected from AIA mice and examined cytokine production by T_(H) cells.In spite of multiple cellular sources of GM-CSF (Cornish et al., 2009).CD4⁺ T_(H) cells were prominent producers of GM-CSF in synovial tissuesof AIA mice (FIG. 28C), consistent with the observation in spleens (FIG.28A). Moreover, a significantly lower percentage of synovialGM-CSF-producing T_(H) cells was detected in Stat5^(−/−) mice thanStat5^(+/+) mice (FIG. 28D). On the other hand, both T_(H)1 and T_(H)17cells exhibited similar percentages between two groups (FIG. 28C). Adecrease in GM-CSF level in synovial compartments of Stat5^(−/−) mice incomparison to controls was expected. To address this, inflamed synovialtissues were harvested from AIA mice for RNA and protein extraction toexamine cytokine level by qPCR and ELISA. Indeed, lower synovial GM-CSFbut not IFN-γ or IL-17 was detected in Stat5^(−/−) mice than Stat5^(+/+)mice at day 5 or 7 after arthritis induction (FIGS. 28E and 30A-30C). Inaddition, two important proinflammatory cytokines IL-6 and IL-1β werealso found persistently and significantly reduced in STAT5-deficientmice (FIGS. 28E and 30A-30C), indicating the attenuated synovialinflammation. Notably. TNF-α production was reduced at day 7 but not atday 5 in STAT5-deficient mice (FIGS. 28E and 30A-C). Together, theseresults indicate that STAT5-regulated GM-CSF expression by arthritogenicT_(H) cells is crucial for evoking synovial inflammation.

To determine the critical role of STAT5-regulated GM-CSF production byT_(H) cells in mediating synovitis and arthritis development, GM-CSF wasadministered via intra-articular injection in mixture with mBSA to theleft knee joints of mBSA/CFA-immunized mice, whereas mBSA was injectedalone to the right knee joints. Injection with mBSA alone was sufficientto induce abundant immune cell infiltration in the synovial compartmentsof Stat5^(+/+) mice but failed to do so in Stat5^(−/−) mice (FIG. 28F).Administration of GM-CSF together with mBSA efficiently restoredsynovial inflammation in Stat5^(−/−) mice (FIG. 34F). Consistently, theSafranin-O/Fast Green staining revealed severe cartilage depletion uponGM-CSF/mBSA injection, but not mBSA alone, in Stat5^(−/−) mice (FIG.28G). These results therefore provide support for the notion thatSTAT5-regulated GM-CSF production by arthritogenic T_(H) cells isessential for mediating arthritis pathogenesis.

Example 13. Th-Cell-Derived GM-CSF Mediates Neutrophil Accumulation inSynovial Tissues

The mechanism by which GM-CSF-producing Th cells evoke synovialinflammation and drive arthritis development was examined. Myeloidlineage-derived cells, including neutrophils, DCs and macrophages,express GM-CSF receptor and are common targets of GM-CSF (Hamilton,2008). Importantly, those cells invade synovial compartments in RApatients and mouse arthritis models, and contribute to synovitis(McInnes and Schett, 2011). The infiltration of myeloid lineage-derivedcells in synovial compartments of ALA mice was examined. CD11b⁺ myeloidcells represented a predominant population (˜70%) among synovialinfiltrating leukocytes (FIG. 31B). Although CD4⁺ T_(H) cellinfiltration was not altered by STAT5 deletion, synovial CD11b⁺ cellinfiltration was significantly reduced in Stat5^(−/−) mice compared withStat5^(+/+) mice when examined at day 7 after arthritis induction (FIG.31B). This reduction is unlikely due to defective hematopoiesis, assimilar frequencies of CD11b⁺ cells were detected in spleens of twogroup (FIG. 31A). Further, CD11b⁺ cells continuously increased insynovial tissues of wild-type mice, but not STAT5-deficient mice, over a7-day time course (FIG. 31C). Notably, the selective ablation ofsynovial CD11b⁺ cell accumulation in STAT5-deficient mice can bepartially restored by local administration of GM-CSF during arthritisinduction (FIG. 31D). Together, these results indicate that myeloid cellaccumulation in synovial compartments may be specifically dampened byT-cell-specific STAT5 deletion and resultant GM-CSF insufficiency.

Next, different populations of CD11b⁺ cells, including DCs, macrophagesand neutrophils were analyzed. Monocyte-derived dendritic cells (MoDCs),characterized as CD11c^(int)CD11b^(hi)Ly6C^(+/hi)MHCII^(hi), wererecently reported to be involved in the mBSA/IL-1β arthritis model(Campbell et al., 2011). In the AIA model of the present study, MoDCswere identified at low abundance in spleens and synovial tissues (datanot shown). Furthermore, comparable frequencies of MoDCs were detectedin both peripheral lymphoid tissues and synovial tissues betweenStat5^(+/+) and Stat5^(−/−) mice (data not shown). These results are inagreement with a previous study showing a dispensable role of GM-CSF inMoDC differentiation (Greter et al., 2012).

Neutrophils have great cytotoxic potential and contribute to the RAinitiation and progression in multiple ways (Wright et al., 2014). Ithas been suggested that RA disease activity and joint destructiondirectly correlates with neutrophil influx to joints (Wright et al.,2014). Based on the differential expression of Ly6C and Ly6G, CD11b⁺myeloid cells can be classified into Ly6C^(lo)Ly6G^(hi) population(neutrophils) and Ly6C^(hi)Ly6G⁻ population (monocytes/macrophages). Thepresent study shows that Ly6C^(lo)Ly6G^(hi) population continued toaccumulate in synovial tissues over a 7-clay time course, andrepresented a predominant population among synovial CD11b⁺ cells inwild-type mice at day 7 after AIA induction, whereas this population waspersistently and dramatically diminished in STAT5-deficient mice (FIG.32A). Using Giemsa stain, it was validated that synovial-infiltratingLy6C^(lo)Ly6G^(hi) population were neutrophils, which displayed typicalpolymorphonuclear characteristics with ring-shaped nuclei (FIG. 32B). Incontrast, synovial-infiltrating Ly6C^(hi)Ly6G⁻ population hadmononuclear morphology and were likely monocytes/macrophages (FIG. 32B).Importantly, intraarticular administration of GM-CSF during arthritisinduction efficiently restored neutrophil accumulation in synovialcompartments of STAT5-deficient mice (FIG. 32C), suggesting a criticalrole of T_(H)-cell-derived GM-CSF in mediating neutrophil accumulationto inflamed joints.

Neutrophils are recruited during inflammation, in which complexinteractions between neutrophils and vascular endothelial cells directneutrophil adhesion and transmigration from circulation to inflamedtissues (Kolaczkowska and Kubes, 2013). In an in vitro transmigrationassay, neutrophil adhesion and migration across monolayers ofendothelial cells was significantly enhanced by GM-CSF aschemoattractant (FIGS. 33A and 33B), suggesting GM-CSF may mediateneutrophil recruitment to inflamed joints in AIA. Effective neutrophilapoptosis is crucial for the resolution of inflammation. However, insynovitis, neutrophil apoptosis is delayed with a result of extendedsurvival and persistent inflammation (Wright et al., 2014). Thus, theeffect of GM-CSF on neutrophil survival was tested and found that GM-CSFhad profound efficacy in delaying neutrophil apoptosis (FIG. 33C).Together, these results indicate that GM-CSF may mediate neutrophilrecruitment and sustain neutrophil survival in synovial compartments andcontribute to persistent synovitis. To determine the critical role ofneutrophils in AIA, \ a neutralizing antibody (1A8) specific for Ly6Gwas used to deplete neutrophils in vivo. The administration ofneutralizing antibody resulted in significant improvement of jointswelling in AIA (FIG. 32D). Thus, neutrophils accumulation mediated byT_(H)-cell-derived GM-CSF are important for AIA development.

Example 14. GM-CSF Enhances Proinflammatory Cytokine Production byMyeloid Cells and Synovial Fibroblasts

Cytokines are important mediators in the cross-talk between innate andadaptive immunity. As shown herein, several proinflammatory cytokines(IL-6, IL-1β and TNF-α), which are in association with RA pathogenesis(Choy and Panayi, 2001), were significantly reduced in synovial tissuesof STAT5-deficient AIA mice (FIGS. 28E and 30A-30C). To gain insightsinto the mechanism underlying the observed cytokine reduction, thecellular sources of these proinflammatory cytokines were examined byfractionating synovial cells into different populations based thedifferential expression of surface markers (FIG. 34A). Cytokine mRNAexpression level in CD45⁺TCRβ⁺ population (T cells), CD45⁺TCRβ⁻CD11c⁻CD11b⁺ population (mostly monocytes/macrophages and neutrophils) andCD45⁺TCRβ⁻ CD11c⁺ population (dendritic cells) was assessed by RT-PCR.GM-CSF, as similar to IL-17 (as a control), was predominantly producedby synovial T cells (FIG. 34B), further reinforcing the importance ofGM-CSF-producing T_(H) cells. In contrast, IL-6 and IL-1β were mainlyproduced by myeloid cells, e.g. CD11b⁺ population and CD11c⁺ population(FIG. 34B). TNF-α was expressed by all three populations, withrelatively lower abundance in T cells (FIG. 34B). Based on thedifferential expression of Ly6C and Ly6G in CD11b⁺ population asdiscussed above, Ly6C^(lo)Ly6G^(hi) population (neutrophils) andLy6C^(hi)Ly6G⁻ population (monocytes/macrophages) were further analyzed,which showed that monocytes/macrophages were likely the major IL-6producers whereas neutrophils seemed to be better producers of IL-1β andTNF-α (FIG. 34C). These results, together with the findings above (FIGS.28E and 30A-30C), indicate a link that T_(H)-cell-secreted GM-CSFelicits proinflammatory cytokine expression from myeloid cells insynovitis.

To test the regulatory role of GM-CSF in the expression of IL-6 andIL-1β, bone marrow-derived macrophages (BMDMs) and bone marrow-deriveddendritic cells (BMDCs) were cultured, and stimulated with GM-CSF.Indeed, GM-CSF stimulation quickly upregulated mRNA expression of bothIL-6 and IL-1β within 1 hour (FIGS. 34D and 34E). In addition, GM-CSFmarkedly increased the secretion of IL-6 from BMDMs in adosage-dependent manner (FIG. 34F), and from BMDCs even at low dosage(FIG. 34G). To induce mature IL-1β secretion, BMDMs were primed with LPSfor 6 h during which different concentrations of GM-CSF was added,followed by ATP stimulation. The addition of GM-CSF significantlyenhanced the secretion of IL-1β into culture supernatant as measured byELISA (FIG. 34H). Synovial fibroblasts, the active players in synovialinflammation (Muller-Ladner et al., 2007), also showed increased IL-1βmRNA expression upon GM-CSF stimulation (FIG. 34I). An inducible effectof GM-CSF on TNF-α expression was not observed in BMDMs, BMDCs orsynovial fibroblasts (data not shown). Given the functional importanceof IL-6 and IL-1β in arthritis development (Choy and Panayi, 2001),T_(H)-cell-secreted GM-CSF may mediate synovial inflammation also viaeliciting the expression of IL- and IL-1β from myeloid cells andsynovial fibroblasts.

TABLE 1 Summary of genes differentially expressed in T_(H)1, T_(H)17,and T_(H)-GM cells Genes differentially Genes differentially expressedin T_(H)1 expressed in T_(H)17 Gene Gene ID Gene Title ID Gene Title10366586 interferon gamma 10353415 interleukin 17F 10598013 chemokine(C-C 10511779 ATPase, H+ motif) receptor 5 /// transporting, chemokine(C-C lysosomal V0 motif) receptor 2 subunit D2 10523717 secreted10345762 interleukin 1 phosphoprotein 1 receptor, type 1 10420308granzyme B 10359697 chemokine (C motif) ligand 1 10545135 interleukin 1210587639 5′ nucleotidase, receptor, beta 2 ecto 10531724placenta-specific 8 10501860 formin binding protein 1-like 10363070glycoprotein 49 A /// 10345032 interleukin 17A leukocyte immunoglobulin-like receptor, subfamily B, member 4 10363082 leukocyte 10446965 RAS,guanyl immunoglobulin- releasing protein 3 like receptor, subfamily B,member 4 10424683 lymphocyte antigen 10565990 ADP- 6 complex, locus Gribosyltransferase 2a 10552406 natural killer cell 10465059 cathepsin Wgroup 7 sequence 10603151 glycoprotein m6b 10358476 proteoglycan 4(megakaryocyte stimulating factor, articular superficial zone protein)10360173 SLAM family 10471953 activin receptor member 7 IIA 10455961interferon inducible 10400006 aryl-hydrocarbon GTPase 1 receptor10400304 EGL nine homolog 10409876 cytotoxic T 3 (C. elegans)lymphocyte- associated protein 2 alpha 10574023 metallothionein 210388591 carboxypeptidase D 10493108 cellular retinoic 10390640 IKAROSfamily acid binding protein zinc finger 3 II 10375436 family with10590623 chemokine (C-X- sequence similarity C motif) receptor 71,member B 6 10398039 serine (or cysteine) 10367734 uronyl-2- peptidaseinhibitor, sulfotransferase clade A, member 3F /// serine (or cysteine)peptidase inhibitor, clade A, member 3G 10349108 serine (or cysteine)10500656 CD101 antigen peptidase inhibitor, clade B, member 5 10607738carbonic anhydrase 10347895 WD repeat 5b, mitochondrial domain 6910496539 guanylate binding 10495854 protease, serine, protein 5 12neurotrypsin (motopsin) 10373918 leukemia inhibitory 10425049apolipoprotein factor L9b /// apolipoprotein L9a 10455954 predicted gene4951 10378286 integrin alpha E, epithelial- associated 10598976 tissueinhibitor of 10362896 CD24a antigen metalloproteinase 1 10492136doublecortin-like 10409866 cytotoxic T kinase 1 lymphocyte- associatedprotein 2 beta 10405211 growth arrest and 10400989 potassium DNA-damage-voltage-gated inducible 45 gamma channel, subfamily H (eag- related),member 5 10503202 chromodomain 10590242 chemokine (C-C helicase DNAmotif) receptor 8 binding protein 7 10542275 ets variant gene 6 10407435aldo-keto (TEL oncogene) reductase family 1, member Cl8 10556820transmembrane 10592023 amyloid beta protein 159 (A4) precursor- likeprotein 2 10444291 histocompatibility 10359480 dynamin 3 2, class IIantigen A, beta 1 10439299 stefin A3 10475544 sema domain, transmembranedomain (TM), and cytoplasmic domain, (semaphorin) 6D 10547641 solutecarrier family 10409767 golgi membrane 2 (facilitated glucose protein 1transporter), member 3 10503200 chromodomain 10392464 family withhelicase DNA sequence binding protein 7 similarity 20, member A 10544320RIKEN cDNA 10504891 transmembrane 1810009J06 gene /// protein with EGF-predicted gene 2663 like and two follistatin-like domains 1 10503218chromodomain 10504817 transforming helicase DNA growth factor, bindingprotein 7 beta receptor 1 10503198 chromodomain 10393559 tissueinhibitor of helicase DNA metalloproteinase binding protein 7 2 10507594solute earner family 10474419 leucine-rich 2 (facilitated glucoserepeat-containing transporter), G protein-coupled member 1 receptor 410438626 ets variant gene 5 10456492 DNA segment, Chr 18, ERATO Doi 653,expressed 10390328 T-box 21 10345241 dystonin 10574027 metallothionein 110471555 angiopoietin-like 2 10493820 S100 calcium 10494821 tetraspanin2 binding protein A6 (calcyclin) 10376324 predicted gene 10542355epithelial 12250 membrane protein 1 10406852 calponin 3, acidic 10500295pleckstrin homology domain containing, family O member 1 10412076 gem(nuclear 10375402 a disintegrin and organelle) metallopeptidaseassociated protein 8 domain 19 (meltrin beta) 10496555 guanylate binding10484227 SEC 14 and protein 1 /// spectrin domains guanylate binding 1protein 5 10345074 centrin 4 10472097 formin-like 2 10503194chromodomain 10587829 procollagen helicase DNA lysine, 2- bindingprotein 7 oxoglutarate 5- dioxygenase 2 10537561 RIKEN cDNA 10530536 tecprotein 1810009J06 gene /// tyrosine kinase predicted gene 2663 10439895activated leukocyte 10586700 RAR-related cell adhesion orphan receptormolecule alpha 10459772 lipase, endothelial 10354191 ring finger protein149 10439762 S- 10438738 B-cell adenosylhomocysteine leukemia/lymphomahydrolase 6 10482030 stomatin 10347888 chemokine (C-C motif) ligand 2010459905 SET binding 10440131 G protein- protein 1 coupled receptor 1510357833 ATPase, Ca++ 10453057 cytochrome P450, transporting, plasmafamily 1, membrane 4 subfamily b, polypeptide 1 /// RIKEN cDNA1700038P13 gene 10475517 expressed sequence 10542140 killer cell lectin-AA467197 /// like receptor microRNA 147 subfamily B member 1F 10585778sema domain, 10471880 microRNA 181b-2 immunoglobulin domain (Ig), andGPI membrane anchor, (semaphorin) 7A 10354529 RIKEN cDNA 10542791 PTPRF1700019D03 gene interacting protein, binding protein 1 (liprin beta 1)10582275 solute carrier family 10583242 sestrin 3 7 (cationic amino acidtransporter, y+ system), member 5 10576034 interferon 10489569phospholipid regulatory factor 8 transfer protein /// cathepsin A10503222 chromodomain 10523297 cyclin G2 helicase DNA binding protein 710503220 chromodomain 10381187 ATPase, H+ helicase DNA transporting,binding protein 7 lysosomal V0 subunit A1 10503210 chromodomain 10346651bone helicase DNA morphogenic binding protein 7 protein receptor, typeII (serine/threonine kinase) 10476945 cystatin F 10490159 prostate(leukocystatin) transmembrane protein, androgen induced 1 10503216chromodomain 10389581 yippee-like 2 helicase DNA (Drosophila) bindingprotein 7 10366983 transmembrane 10581992 avian protein 194musculoaponeurotic fibrosarcoma (v-maf) AS42 oncogene homolog 10495675coagulation factor 10413250 cytoplasmic III polyadenylated homeobox10421697 RIKEN cDNA 10555063 integrator 9030625A04 gene complex subunit4 10445112 ubiquitin D 10406982 a disintegrin-like and metallopeptidase(reprolysin type) with thrombospondin type 1 motif, 6 10530627 leucinerich repeat 10596303 acid phosphatase, containing 66 prostate 10440019transmembrane 10357472 chemokine (C-X- protein 45a C motif) receptor 410378783 ribosomal protein 10545130 growth arrest and L36 DNA-damage-inducible 45 alpha 10447341 ras homolog gene 10436402 claudin domainfamily, member Q /// containing 1 phosphatidylinositol glycan anchorbiosynthesis, class F 10373452 predicted gene 129 10539135 cappingprotein (actin filament), gelsolin-like 10454286 microtubule- 10428534trichorhinophalangeal associated protein, syndrome 1 RP/EB family,(human) member 2 10572497 interleukin 12 10368675 myristoylatedreceptor, beta 1 alanine rich protein kinase C substrate 10368060epithelial cell 10531910 hydroxysteroid transforming (17-beta) sequence2 dehydrogenase 13 oncogene-like 10471457 ST6 (alpha-N- 10370303adenosine acetyl-neuraminyl- deaminase, RNA- 2,3-beta-galactosyl-specific, B1 1,3)-N- acetylgalactosaminide alpha-2,6- sialyltransferase4 10374366 epidermal growth 10592888 chemochine (C- factor receptor X-Cmotif) receptor 5 10450501 tumor necrosis 10503259 transformation factorrelated protein 53 inducible nuclear protein 1 10347291 chemokine (C-X-C10446771 lysocardiolipin motif) receptor 2 acyltransferase 1 10553501solute carrier family 10428579 exostoses 17 (sodium- (multiple) 1dependent inorganic phosphate cotransporter), member 6 10345824interleukin 18 10476314 prion protein receptor accessory protein10458314 transmembrane 10406598 serine protein 173 incorporator 510388430 serine (or cysteine) 10461765 leupaxin peptidase inhibitor,clade F, member 1 10496015 phospholipase A2, 10428536trichorhinophalangeal group XIIA syndrome 1 (human) 10510391 spermidinesynthase 10362245 erythrocyte protein band 4.1- like 2 10486396EH-domain 10604008 predicted gene containing 4 10058 /// predicted gene10230 /// predicted gene 10486 /// predicted gene 14632 /// predictedgene 14819 /// predicted gene 4836 /// predicted gene 2012 /// predictedgene 5169 /// predicted gene 6121 /// Sycp3 like X-linked /// predictedgene 5168 /// predicted gene 10488 /// predicted gene 14525 ///predicted gene 5935 10368054 epithelial cell 10409857 RIKEN cDNAtransforming 4930486L24 gene sequence 2 oncogene-like 10608637 na10522368 NIPA-like domain containing 1 10595718 carbohydrate 10368720solute carrier sulfotransferase 2 family 16 (monocarboxylic acidtransporters), member 10 10496580 guanylate binding 10438639diacylglycerol protein 3 kinase, gamma 10594053 promyelocytic 10499431synaptotagmin XI leukemia 10544829 JAZF zinc finger 1 10565840neuraminidase 3 10601778 armadillo repeat 10494023 RAR-relatedcontaining, X-linked orphan receptor 3 gamma 10355967 adaptor-related10391103 junction protein complex AP- plakoglobin 1, sigma 3 10592503cytotoxic and 10417053 muscleblind-like regulatory T cell 2 molecule10496023 caspase 6 10350341 microRNA 181b-1 10599192 LON peptidase N-10459071 RIKEN cDNA terminal domain and 2010002N04 gene ring finger 310467578 phosphoinositide-3- 10463476 Kazal-type serine kinase adaptorpeptidase inhibitor protein 1 domain 1 10585703 ribonuclease P 2510348537 receptor subunit (human) (calcitonin) activity modifyingprotein 1 10365482 tissue inhibitor of 10348432 ArfGAP withmetalloproteinase 3 GTPase domain, ankyrin repeat and PH domain 110469151 inter-alpha 10576332 tubulin, beta 3 /// (globulin) inhibitormelanocortin 1 H5 receptor 10503192 chromodomain 10554094 insulin-likehelicase DNA growth factor 1 binding protein 7 receptor 10593050interleukin 10 10495794 phosphodiesterase receptor, alpha 5A, cGMP-specific 10597648 myeloid 10569504 tumor necrosis differentiation factorreceptor primary response superfamily, gene 88 member 23 10538290sorting nexin 10 10452516 ankyrin repeat domain 12 10503204 chromodomain10534596 cut-like helicase DNA homeobox 1 binding protein 7 10353707protein tyrosine 10362073 serum/glucocorticoid phosphatase 4a1 ///regulated protein tyrosine kinase 1 phosphatase 4a1- like 10377010 SCOcytochrome 10408331 acyl-CoA oxidase deficient thioesterase 13 homolog 1(yeast) 10440903 RIKEN cDNA 10415413 NYN domain and 4932438H23 generetroviral integrase containing 10521205 SH3-domain 10598359synaptophysin binding protein 2 10604587 microRNA 363 10544114homeodomain interacting protein kinase 2 10571958 SH3 domain 10436128myosin, heavy- containing ring chain 15 finger 1 10357553 interleukin 2410408450 SRY-box containing gene 4 10606730 armadillo repeat 10487011glycine containing, X-linked amidinotransferase 6 (L- arginine: glycineamidinotransferase) 10564960 furin (paired basic 10378833 slingshotamino acid cleaving homolog 2 enzyme) (Drosophila) 10402585tryptophanyl-tRNA 10521498 collapsin synthetase response mediatorprotein 1 10417095 FERM, RhoGEF 10538939 eukaryotic (Arhgef) andtranslation pleckstrin domain initiation factor 2 protein 1 alpha kinase3 (chondrocyte- derived) 10442435 ribonucleic acid 10585276 POU domain,binding protein S1 class 2, associating factor 1 10394990 membrane bound10512156 aquaporin 3 O-acyltransferase domain containing 2 10538753atonal homolog 1 10469110 USP6 N-terminal (Drosophila) like 10351667signaling 10568392 regulator of G- lymphocytic protein signallingactivation molecule 10 family member 1 10461844 guanine nucleotide10603346 proteolipid binding protein, protein 2 alpha q polypeptide10422057 ribosomal protein 10353947 transmembrane L7A protein 13110572897 heme oxygenase 10452633 TGFB-induced (decycling) 1 factorhomeobox 1 10507784 palmitoyl-protein 10380289 monocyte to thioesterase1 macrophage differentiation- associated 10445702 ubiquitin specific10521969 IMP1 inner peptidase 49 mitochondrial membrane peptidase-like(S. cerevisiae) 10569057 ribonuclease/ 10521678 CD38 antigen angiogenininhibitor 1 10370471 1-acylglycerol-3- 10592515 ubiquitin phosphate O-associated and acyltransferase 3 SH3 domain containing, B 10586591carbonic 10512470 CD72 antigen anyhydrase 12 10512701 translocase ofouter 10587085 cDNA sequence mitochondrial BC031353 membrane 5 homolog(yeast) 10462702 HECT domain 10492689 platelet-derived containing 2growth factor, C polypeptide 10552740 nucleoporin 62 /// 10514221perilipin 2 Nup62-Il4i1 protein 10581996 chromodomain 10458247 leucinerich protein, Y repeat chromosome-like 2 transmembrane neuronal 210363901 ets variant gene 5 10468898 lymphocyte transmembrane adaptor 110520862 fos-like antigen 2 10555059 potassium channel tetramerisationdomain containing 14 10526520 procollagen-lysine, 10408629 RIKEN cDNA2-oxoglutarate 5- 1300014106 gene dioxygenase 3 10571274 glutathione10546510 leucine-rich reductase repeats and immunoglobulin- like domains1 10351206 selectin, platelet 10544596 transmembrane protein 176B10493474 mucin 1, 10361748 F-box protein 30 transmembrane 10370000glutathione S- 10356291 RIKEN cDNA transferase, theta 1 A530040E14 gene10500272 predicted gene 129 10581450 DEAD (Asp-Glu- Ala-Asp) boxpolypeptide 28 10452815 xanthine 10414417 pellino 2 dehydrogenase10393823 prolyl 4- 10372528 potassium large hydroxylase, betaconductance polypeptide calcium-activated channel, subfamily M, betamember 4 /// RIKEN cDNA 1700058G18 gene 10408280 leucine rich repeat10408613 tubulin, beta 2B containing 16A 10575685 nudix (nucleoside10411274 synaptic vesicle diphosphate linked glycoprotein 2c moietyX)-type motif 7 10599174 interleukin 13 10456357 phorbol-12- receptor,alpha 1 myristate-13- acetate-induced protein 1 10458940 zinc fingerprotein 10511498 pleckstrin 608 homology domain containing, family F(with FYVE domain) member 2 10476197 inosine 10402136 G protein-triphosphatase coupled receptor (nucleoside 68 triphosphatepyrophosphatase) 10419790 ajuba 10549990 vomeronasal 1 receptor, G10 ///vomernasal 1 receptor Vmn1r- ps4 /// vomeronasal 1 receptor 3 ///vomeronasal 1 receptor Vmn1r238 /// vomeronasal 1 receptor 2 10364909ornithine 10554789 cathepsin C decarboxylase antizyme 1 /// ornithinedecarboxylase antizyme 1 pseudogene 10503190 chromodomain 10427928triple functional helicase DNA domain (PTPRF binding protein 7interacting) 10516932 sestrin 2 10549162 ST8 alpha N acetyl- neuraminidealpha-2,8- sialyltransferase 1 10585338 KDEL (Lys-Asp- 10482109mitochondrial Glu-Leu) containing ribosome 2 recycling factor /// RNAbinding motif protein 18 10464425 G protein-coupled 10425092 cytohesin 4receptor kinase 5 10441601 T-cell activation 10356866 programmed cellRho GTPase- death 1 activating protein 10482059 glycoprotein 10554204ATP/GTP galactosyltransferase binding protein- alpha 1,3 like 1 10522411cell wall biogenesis 10403229 integrin beta 8 43 C-terminal homolog (S.cerevisiae) 10369276 coiled-coil domain 10374529 expressed containing109A sequence AV249152 10368970 PR domain 10565434 ribosomal proteincontaining 1, with S13 ZNF domain 10369541 hexokinase 1 10431266ceramide kinase 10374236 uridine 10410124 cathepsin L phosphorylase 110489660 engulfment and cell 10441003 runt related motility 2, ced-12transcription homolog factor 1 (C. elegans) 10488797 peroxisomal10555303 phosphoglucomutase membrane protein 4 2-like 1 10558090transforming, acidic 10530215 RIKEN cDNA coiled-coil 1110003E01 genecontaining protein 2 10409265 AU RNA binding 10480275 nebuletteprotein/enoyl- coenzyme A hydratase 10374364 thymoma viral 10434302kelch-like 24 proto-oncogene 2 (Drosophila) 10598575 LanC (antibiotic10565002 CREB regulated synthetase transcription component C-like 3coactivator 3 (bacterial) 10439514 growth associated 10413338 na protein43 10497842 Bardet-Biedl 10523670 AF4/FMR2 syndrome 7 (human) family,member 1 10462091 Kruppel-like factor 10478594 cathepsin A 9 ///predicted gene 9971 10498024 solute carrier family 10514128tetratricopeptide 7 (cationic amino repeat domain acid transporter, y+39B system), member 11 10483719 chimerin 10535956 StAR-related(chimaerin) 1 lipid transfer (START) domain containing 13 10606694Bruton 10503695 BTB and CNC agammaglobulinemia homology 2 tyrosinekinase 10443110 synaptic Ras 10584334 sialic acid GTPase activatingacetylesterase protein 1 homolog (rat) 10368062 epithelial cell 10502890ST6 (alpha-N- transforming acetyl- sequence 2 neuraminyl-2,3-oncogene-like beta-galactosyl- 1,3)-N- acetylgalactosaminide alpha-2,6-sialyltransferase 3 10575693 vesicle amine 10564467 leucine richtransport protein 1 repeat containing homolog-like 28 (T. californica)10562897 zinc finger protein 10345715 mitogen-activated 473 /// vacciniaprotein kinase related kinase 3 kinase kinase kinase 4 10373709eukaryotic 10568668 a disintegrin and translation initiationmetallopeptidase factor 4E nuclear domain 12 import factor 1 (meltrinalpha) 10487238 histidine 10462406 RIKEN cDNA decarboxylase C030046E11gene 10594988 mitogen-activated 10472649 myosin IIIB protein kinase 610422436 dedicator of 10363894 inositol cytokinesis 9 polyphosphatemultikinase 10459084 synaptopodin 10606058 chemokine (C-X- C motif)receptor 3 10567450 dynein, axonemal, 10439955 family with heavy chain 3sequence similarity 55, member C 10604751 fibroblast growth 10530615OC1A domain factor 13 containing 2 10584827 myelin protein 10528183spermatogenesis zero-like 2 associated glutamate (E)-rich protein 4d ///spermatogenesis associated glutamate (E)-rich protein 4c ///spermatogenesis associated glutamate (E)-rich protein 4e /// predictedgene 9758 /// RIKEN cDNA 4930572O03 gene /// spermatogenesis associatedglutamate (E)-rich protein 7, pseudogene 1 /// predicted gene 736110473356 ubiquitin- 10488507 abhydrolase conjugating enzyme domaincontaining E2L6 12 10498350 purinergic receptor 10420668 microRNA 15aP2Y, G-protein coupled, 14 10497862 transient receptor 10469951 ringfinger potential cation protein 208 channel, subfamily C, member 310368056 epithelial cell 10501629 CDC14 cell transforming division cycle14 sequence 2 homolog A oncogene-like (S. cerevisiae) 10425357Smith-Magenis 10386789 Unc-51 like syndrome kinase 2 chromosome region,(C. elegans) candidate 7-like (human) 10498952 guanylate cyclase 1,10401138 ATPase, H+ soluble, alpha 3 transporting, lysosomal V1 subunitD 10548905 epidermal growth 10554118 family with factor receptorsequence pathway substrate 8 similarity 169, member B 10579703 calcium10603843 synapsin I homeostasis endoplasmic reticulum protein /// RIKENcDNA 1700030K09 gene 10404630 RIO kinase 1 10575184 WW domain (yeast)containing E3 ubiquitin protein ligase 2 10518069 EF hand domain10537712 glutathione S- containing 2 transferase kappa 1 10469672glutamic acid 10511541 dpy-19-like 4 decarboxylase 2 (C. elegans)10526941 RIKEN cDNA 10394816 predicted gene D830046C22 gene 928210567448 dynein, axonemal, 10587503 SH3 domain heavy chain 3 bindingglutamic acid-rich protein like 2 10437885 myosin, heavy 10411359proteolipid polypeptide 11, protein 2 smooth muscle 10600122 X-linked10579939 ubiquitin specific lymphocyte- peptidase 38 /// regulated 3B/// X- predicted gene linked lymphocyte- 9725 regulated 3C /// X- linkedlymphocyte- regulated 3A 10587665 RIKEN cDNA 10370242 poly(rC) binding4930579C12 gene protein 3 10350753 glutamate- ammonia ligase (glutaminesynthetase) 10456296 mucosa associated lymphoid tissue lymphomatranslocation gene 1 10380571 guanine nucleotide binding protein (Gprotein), gamma transducing activity polypeptide 2 /// ABI gene family,member 3 10369413 sphingosine phosphate lyase 1 10552276 ubiquitin-conjugating enzyme E2H /// predicted gene 2058 10394532 ubiquitin-conjugating enzyme E2F (putative) /// ubiquitin- conjugating enzyme E2F(putative) pseudogene 10556463 aryl hydrocarbon receptor nucleartranslocator-like 10471994 kinesin family member 5C 10395328 sortingnexin 13 10599348 glutamate receptor, ionotropic, AMPA3 (alpha 3)10601595 RIKEN cDNA 3110007F17 gene /// predicted gene 6604 ///predicted gene 5167 /// predicted gene 2411 /// predicted gene 1495710372891 SLIT-ROBO Rho GTPase activating protein 1 10355024 islet cellautoantigen 1-like 10518147 podoplanin 10473537 olfactory receptor 112310424411 tumor susceptibility gene 101 10439960 centrosomal protein 9710551852 CAP-GLY domain containing linker protein 3 10599291reproductive homeobox 4E /// reproductive homeobox 4G /// reproductivehomeobox 4F /// reproductive homeobox 4A /// reproductive homeobox 4C/// reproductive homeobox 4B /// reproductive homeobox 4D 10587315glutathione S- transferase, alpha 4 10447167 metastasis associated 310480288 nebulette 10491300 SKl-like 10596637 mitogen-activated proteinkinase- activated protein kinase 3 10518019 DNA-damage inducible protein2 /// regulatory solute carrier protein, family 1, member 1 10384685RIKEN cDNA 1700093K21 gene (0439483 Rho GTPase activating protein 3110353844 neuralized homolog 3 homolog (Drosophila) 10459604 RIKEN cDNA4933403F05 gene 10488892 transient receptor potential cation channel,subfamily C, member 4 associated protein 10542822 RAB15 effector protein10553354 neuron navigator 2 10425966 ataxin 10 10360506 thymoma viralproto-oncogene 3 10531610 RasGEF domain family, member 1B 10417787guanine nucleotide binding protein (G protein), gamma 2 10381588granulin 10437080 tetratricopeptide repeat domain 3 10509560 ribosomalprotein L38 10466886 na 10580457 NEDD4 binding protein 1 10451061 runtrelated transcription factor 2 10433953 yippee-like 1 (Drosophila)10447461 stonin 1 10501909 methyltransferase like 14 /// Sec24 relatedgene family, member D (S. cerevisiae) 10519693 sema domain,immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin)3D 10385557 CCR4-NOT transcription complex, subunit 6 10413047plasminogen activator, urokinase 10406663 arylsulfatase B 10430113 RhoGTPase activating protein 39 10475830 mitochondrial ribosomal protein S510410892 RAS p21 protein activator 1 10515994 stromal membrane-associated GTPase-activating protein 2 10410099 CDC14 cell divisioncycle 14 homolog B (S. cerevisiae) 10428157 ring finger protein 19A10563643 tumor susceptibility gene 101 10412260 follistatin /// thyroidhormone receptor associated protein 3 10386539 similar to ubiquitin A-52residue ribosomal protein fusion product 1 10415574 cyclin 1 10494978protein tyrosine phosphatase, non- receptor type 22 (lymphoid) 10511416thymocyte selection- associated high mobility group box 10562500dpy-19-like 3 (C. elegans) 10568135 proline-rich transmembrane protein 2/// RIKEN cDNA 2900092E17 gene 10514466 Jun oncogene 10500847 membraneassociated guanylate kinase, WW and PDZ domain containing 3 10549760zinc finger protein 580 10549377 RIKEN cDNA 1700034J05 gene 10430174apolipoprotein L9a /// apolipoprotein L9b 10474333 elongation protein 4homolog (S. cerevisiae) 10560791 predicted gene, EG381936 /// predictedgene 6176 10407159 ankyrin repeat domain 55 10603659 mediator complexsubunit 14 10576854 cortexin 1 10353775 BEN domain containing 6 10573865predicted gene 3579 10356886 solute carrier organic anion transporterfamily, member 4C1 10507273 phosphatidylinositol 3 kinase, regulatorysubunit, polypeptide 3 (p55) 10424252 WDYHV motif containing 1 10518735splA/ryanodine receptor domain and SOCS box containing 1 10562576pleckstrin homology domain containing, family F (with FYVE domain)member 1 10375667 ring finger protein 130 10528268 protein tyrosinephosphatase, non- receptor type 12 10593205 REX2, RNA exonuclease 2homolog (S. cerevisiae) 10576056 microtubule- associated protein 1 lightchain 3 beta 10547916 parathymosin 10377689 gamma- aminobutyric acidreceptor associated protein 10602307 ovary testis transcribed ///predicted gene 15085 /// predicted gene 15127 /// predicted gene,OTTMUSG00000019001 /// leucine zipper protein 4 /// predicted gene 15097/// predicted gene 15091 /// predicted gene 10439 /// predicted gene15128 10426835 DIP2 disco- interacting protein 2 homolog B (Drosophila)10439798 DAZ interacting protein 3, zinc finger 10375614 glutaminefructose-6- phosphate transaminase 2 10361882 NHS-like 1 10419274 gliamaturation factor, beta 10424781 glutamate receptor, ionotropic, N-methyl D- aspartate- associated protein 1 (glutamate binding) 10546960na 10514713 WD repeat domain 78 10394954 grainyhead-like 1 (Drosophila)10437205 Purkinje cell protein 4 10464251 attractin like 1 10496251 3-hydroxybutyrate dehydrogenase, type 2 10396383 solute carrier family 38,member 6 10585794 cytochrome P450, family 11, subfamily a, polypeptide 110385719 Sec24 related gene family, member A (S. cerevisiae) 10407358polyadenylate binding protein- interacting protein 1 10498775 golgiintegral membrane protein 4 10584435 von Willebrand factor A domaincontaining 5A 10466304 deltex 4 homolog (Drosophila) 10598292 forkheadbox P3 /// RIKEN cDNA 4930524L23 gene /// coiled-coil domain containing22 10472440 Taxi (human T- cell leukemia virus type I) binding protein 310398455 protein phosphatase 2, regulatory subunit B (B56), gammaisoform 10493076 SH2 domain protein 2A 10409152 RIKEN cDNA 1110007C09gene 10542880 RIKEN cDNA 4833442J19 gene 10378523 Smg-6 homolog,nonsense mediated mRNA decay factor (C. elegans) 10531560 anthrax toxinreceptor 2 10467319 retinol binding protein 4, plasma 10395978 predictedgene 527 10471715 mitochondrial ribosome recycling factor 10511755 WWdomain containing E3 ubiquitin protein ligase 1 10353754 zinc fingerprotein 451 10477572 chromatin modifying protein 4B 10359161 sterol O-acyltransferase 1 10462035 lactate dehydrogenase B 10543319 family withsequence similarity 3, member C 10579052 predicted gene 10033 10475532sulfide quinone reductase-like (yeast) 10428857 metastasis suppressor 110475144 calpain 3 /// glucosidase, alpha; neutral C 10396645 zincfinger and BTB domain containing 1 10428302 Kruppel-like factor 1010577882 heparan-alpha- glucosaminide N- acetyltransferase 10548069dual-specificity tyrosine-(Y)- phosphorylation regulated kinase 410436053 developmental pluripotency associated 2 10401564 RIKEN cDNA1110018G07 gene 10471535 family with sequence similarity 129, member B10349404 mannoside acetylglucosaminyltransferase 5 10520173 amiloride-sensitive cation channel 3 10508860 solute carrier family 9(sodium/hydrogen exchanger), member 1 10374500 vacuolar protein sorting54 (yeast) 10387723 RIKEN cDNA 2810408A11 gene 10488020 thioredoxin-related transmembrane protein 4 10411126 junction- mediating andregulatory protein 10345706 DNA segment, Chr 1, Brigham & WomensGenetics 0212 expressed 10364375 cystatin B 10480379 mitochondrialribosomal protein S5 10521243 G protein- coupled receptor kinase 410497920 ankyrin repeat domain 50 10593723 acyl-CoA synthetase bubblegumfamily member 1 10375634 mitogen-activated protein kinase 9 10384555aftiphilin 10468113 Kv channel- interacting protein 2 10423363progressive ankylosis 10538150 transmembrane protein 176A 10396485synaptic nuclear envelope 2 10401007 protein phosphatase 2, regulatorysubunit B (B56), epsilon isoform 10419151 eosinophil- associated,ribonuclease A family, member 1 10390768 SWI/SNF related, matrixassociated, actin dependent regulator of chromatin, subfamily e, member1 10478145 protein phosphatase 1, regulatory (inhibitor) subunit 16B10433057 calcium binding and coiled coil domain 1 10545921 MAXdimerization protein 1 10392449 WD repeat domain, phosphoinositideinteracting 1 10545608 sema domain, immunoglobulin domain (Ig), TMdomain, and short cytoplasmic domain 10567219 ADP-ribosylationfactor-like 6 interacting protein 1 10471201 c-abl oncogene 1, receptortyrosine kinase 10505841 predicted gene 13271 /// predicted gene 13290/// predicted gene 13277 /// predicted gene 13276 10414360 lectin,galactose binding, soluble 3 10403258 guanosine diphosphate (GDP)dissociation inhibitor 2 10476759 Ras and Rab interactor 2 10430866cytochrome P450, family 2, subfamily d, polypeptide 10 10432619 POUdomain, class 6, transcription factor 1 10521972 protocadherin 710350646 ER degradation enhancer, mannosidase alpha-like 3 10440993regulator of calcineurin 1 10505008 solute carrier family 44, member 110566670 olfactory receptor 478 10356172 phosphotyrosine interactiondomain containing 1 10418506 stabilin 1 10419429 olfactory receptor 723/// olfactory receptor 724 10581434 dipeptidase 2 10401365 zinc finger,FYVE domain containing 1 10591188 olfactory receptor 843 10565846 signalpeptidase complex subunit 2 homolog (S. cerevisiae) 10467258 myoferlin10548547 predicted gene 6600 10523012 deoxycytidine kinase 10348547ubiquitin- conjugating enzyme E2F (putative) 10483667 corepressorinteracting with RBPJ, 1 10584071 PR domain containing 10 10585249protein phosphatase 2 (formerly 2A), regulatory subunit A (PR 65), betaisoform 10546137 ankyrin repeat and BTB (POZ) domain containing 110484720 olfactory receptor 1166 10571415 vacuolar protein sorting 37 A(yeast) 10595189 solute carrier family 17 (anion/sugar transporter),member 5 10584426 olfactory receptor 910 10585986 myosin IXa 10401753VPS33B interacting protein, apical- basolateral polarity regulator10349793 dual serine/threonine and tyrosine protein kinase 10527528SWI/SNF related, matrix associated, actin dependent regulator ofchromatin, subfamily e, member 1 10485767 olfactory receptor 127710557459 mitogen-activated protein kinase 3 10471486 endoglin 10420846frizzled homolog 3 (Drosophila) 10405849 olfactory receptor 466 10568691RIKEN cDNA A130023I24 gene 10351111 dynamin 3, opposite strand ///microRNA 214 /// microRNA 199a-2 10540785 RIKEN cDNA 6720456B07 gene10540923 makorin, ring finger protein, 2 10413416 interleukin 17receptor D 10386636 ubiquitin specific peptidase 22 10383799transcobalamin 2 Genes differentially Genes upregulated Genesdownregulated on upregulated in T_(H)-GM cells on T_(H)-GM surfaceT_(H)-GM cells surface Gene Gene Gene ID Gene Title ID Gene Title SymbolGene Title 10385918 interleukin 3 10435704 CD80 antigen Ly6a lymphocyteantigen 6 complex, locus A 10511363 preproenkephalin 10548409 killercell lectin- Cd27 CD27 antigen like receptor subfamily C, member 110497878 interleukin 2 10421737 tumor necrosis Sell selectin, factor(ligand) lymphocyte superfamily, member 11 10385912 colony 10597420chemokine (C-C Ctsw cathepsin W stimulating factor motif) receptor 4 2(granulocyte- macrophage) 10404422 serine (or 10441633 chemokine (C-CLtb lymphotoxin B cysteine) motif) receptor 6 peptidase inhibitor, cladeB, member 6b 10408689 neuritin 1 10365933 early endosome Gngt2 guaninenucleotide antigen 1 binding protein (G protein), gamma transducingactivity polypeptide 2 /// ABI gene family, member 3 10467979 stearoyl-10404840 CD83 antigen Gpr18 G protein-coupled Coenzyme A receptor 18desaturase 1 10469312 phosphotriesterase 10359434 Fas ligand (TNF Igfbp4insulin-like growth related /// superfamily, factor binding Clq-like 3member 6) protein 4 10435704 CD80 antigen 10344966 lymphocyte Il17rainterleukin 17 antigen 96 receptor A 10502655 cysteine rich 10345752interleukin 1 Il18r1 interleukin 18 protein 61 receptor, type IIreceptor 1 10350159 ladinin 10439527 T cell Klrd1 killer cell lectin-immunoreceptor like receptor, with Ig and ITIM subfamily D, domainsmember 1 10548409 killer ceil lectin- 10494595 Notch gene Mctp2 multipleC2 like receptor homolog 2 domains, subfamily C, (Drosophila)transmembrane 2 member 1 10571399 zinc finger, 10597279 chemokine (C-CMs4a6b membrane- DHHC domain motif) receptor- spanning 4- containing 2like 2 domains, subfamily A, member 6B 10538791 TNFAIP3 10485405 CD44antigen Pld3 phospholipase D interacting family, member 3 protein 310407126 polo-like kinase 10561104 AXL receptor Pyhin1 pyrin and HIN 2(Drosophila) tyrosine kinase domain family, member 1 10355984 serine (or10585048 cell adhesion S1pr1 sphingosine-1- cysteine) molecule 1phosphate receptor peptidase 1 inhibitor, clade E, member 2 10421737tumor necrosis Slc44a2 solute carrier factor (ligand) family 44, membersuperfamily, 2 member 11 10503023 cystathionase (cystathioninegamma-lyase) 10389207 chemokine (C-C motif) ligand 5 10361887 PERP, TP53apoptosis effector 10530841 insulin-like growth factor binding protein 710504838 nuclear receptor subfamily 4, group A, member 3 10482762isopentenyl- diphosphate delta isomerase 10597420 chemokine (C-C motif)receptor 4 10441633 chemokine (C-C motif) receptor 6 10595402 familywith sequence similarity 46, member A 10480139 Clq-like 3 ///phosphotriesterase related 10540472 basic helix-loop- helix family,member e40 10404429 serine (or cysteine) peptidase inhibitor, clade B,member 9 10595404 family with sequence similarity 46, member A 10365933early endosome antigen 1 10384373 fidgetin-like 1 10400072 scinderin10377938 enolase 3, beta muscle 10589994 eomesodermin homolog (Xenopuslaevis) 10404840 CD83 antigen 10485624 proline rich Gla (G-carboxyglutamic acid) 4 (transmembrane) 10369102 predicted gene 976610505030 fibronectin type III and SPRY domain containing 1-like 10606868brain expressed gene 1 10501832 ATP-binding cassette, sub- family D(ALD), member 3 10457225 mitogen- activated protein kinase kinase kinase8 10554521 phosphodiesterase 8A 10446229 tumor necrosis factor (ligand)superfamily, member 9 10593842 tetraspanin 3 10407211 phosphatidic acidphosphatase type 2A 10488655 BCL2-like 1 10470182 brain expressedmyelocytomatosis oncogene 10445977 Epstein-Barr virus induced gene 310587495 interleukin-1 receptor- associated kinase 1 binding protein 110419082 RIKEN cDNA 5730469M10 gene 10472212 plakophilin 4 10487588interleukin 1 alpha 10359434 Fas ligand (TNF superfamily, member 6)10351015 serine (or cysteine) peptidase inhibitor, clade C(antithrombin), member 1 10344966 lymphocyte antigen 96 10488415cystatin C 10598771 monoamine oxidase A 10345752 interleukin 1 receptor,type II 10588577 cytokine inducible SH2- containing protein 10439527 Tcell immunoreceptor with Ig and ITIM domains 10511258 family withsequence similarity 132, member A 10403584 nidogen 1 10399973 histonedeacetylase 9 10494595 Notch gene homolog 2 (Drosophila) 10346168 signaltransducer and activator of transcription 4 10350630 family withsequence similarity 129, member A 10564667 neurotrophic tyrosine kinase,receptor, type 3 10419288 GTP cyclohydrolase 1 10407535 ribosomalprotein L10A /// ribosomal protein L10A, pseudogene 2 10468945acyl-Coenzyme A binding domain containing 7 10435271 HEG homolog 1(zebrafish) 10576639 neuropilin 1 10505059 T-cell acute lymphocyticleukemia 2 10457091 neuropilin (NRP) and tolloid (TLL)- like 1 10428081heat-responsive protein 12 10435712 CD80 antigen 10597279 chemokine (C-Cmotif) receptor- like 2 10485405 CD44 antigen 10436662 microRNA 15510562044 zinc finger and BTB domain containing 32 10463599 nuclearfactor of kappa light polypeptide gene enhancer in B- cells 2, p49/p10010456005 CD74 antigen (invariant polypeptide of major histocompatibilitycomplex, class II antigen- associated) 10490903 carbonic anhydrase 1310468762 RIKEN cDNA 4930506M07 gene 10470316 na 10363195 heat shockfactor 2 10596652 HemK methyltransferase family member 1 10435693cytochrome c oxidase, subunit XVII assembly protein homolog (yeast)10544660 oxysterol binding protein- like 3 10384725reticuloendotheliosis oncogene 10408600 serine (or cysteine) peptidaseinhibitor, clade B, member 6a 10391444 RUN domain containing 1 /// RIKENcDNA 1700113I22 gene 10561516 nuclear factor of kappa light polypeptidegene enhancer in B- cells inhibitor, beta 10566846 DENN/MADD domaincontaining 5A 10435048 Tctex1 domain containing 2 10470175 lipocalin 1310586250 DENN/MADD domain containing 4A 10512774 coronin, actin bindingprotein 2A 10366546 carboxypeptidase M 10354286 KDEL (Lys- Asp-Glu-Leu)containing 1 10547621 apolipoprotein B mRNA editing enzyme, catalyticpolypeptide 1 10440419 B-cell translocation gene 3 /// B-celltranslocation gene 3 pseudogene 10407467 aldo-keto reductase family 1,member E1 10558580 undifferentiated embryonic cell transcription factor1 10544644 na 10424543 WNT1 inducible signaling pathway protein 110507137 PDZK1 interacting protein 1 10384691 RIKEN cDNA 0610010F05 gene10565315 fumarylacetoacetate hydrolase 10586248 DENN/MADD domaincontaining 4A 10561104 AXL receptor tyrosine kinase 10385837 interleukin13 10440393 SAM domain, SH3 domain and nuclear localization signals, 110401987 potassium channel, subfamily K, member 10 10453715 RAB18,member RAS oncogene family 10496466 alcohol dehydrogenase 4 (class II),pipolypeptide 10396712 fucosyltransferase 8 10603708 calcium/calmodulin-dependent serine protein kinase (MAGUK family) 10352178 saccharopinedehydrogenase (putative) /// similar to Saccharopine dehydrogenase(putative) 10349081 PH domain and leucine rich repeat proteinphosphatase 1 10364950 growth arrest and DNA- damage- inducible 45 beta10566877 SET binding factor 2 10575160 nuclear factor of activatedT-cells 5 10458090 receptor accessory protein 5 10439845 predicted gene5486 10461558 solute carrier family 15, member 3 10586254 DENN/MADDdomain containing 4A 10574166 copine II 10598467 proviral integrationsite 2 10447084 galactose mutarotase 10366346 pleckstrin homology-likedomain, family A, member 1 10355567 transmembrane BAX inhibitor motifcontaining 1 10407420 neuroepithelial cell transforming gene 1 10411882neurolysin (metallopeptidase M3 family) 10585048 cell adhesion molecule1 10538890 hypothetical protein LOC641050 10406681 adaptor-relatedprotein complex 3, beta 1 subunit 10455647 tumor necrosis factor, alpha-induced protein 8 10447521 transcription factor B1, mitochondrial ///T-cell lymphoma invasion and metastasis 2 10523772 leucine rich repeatcontaining 8D 10417759 ubiquitin- conjugating enzyme E2E 2 (UBC4/5homolog, yeast) 10586244 DENN/MADD domain containing 4A 10436500 glucan(1,4- alpha), branching enzyme 1 10556297 adrenomedullin 10593492 zincfinger CCCH type containing 12C 10373358 interleukin 23, alpha subunitp19 10358583 hemicentin 1 10567995 nuclear protein 1 10512030 RIKEN cDNA3110043021 gene 10594652 lactamase, beta 10344960 transmembrane protein70 10399908 protein kinase, cAMP dependent regulatory, type II beta10605766 melanoma antigen, family D, 1 10474141 solute carrier family 1(glial high affinity glutamate transporter), member 2 10461909 cDNAsequence BC016495 10548030 CD9 antigen 10525473 transmembrane protein120B 10435266 HEG homolog 1 (zebrafish) 10593483 ferredoxin 1 10476569RIKEN cDNA 2310003L22 gene 10526718 sperm motility kinase 3A /// spermmotility kinase 3B /// sperm motility kinase 3C 10547613 ribosomalmodification protein rimK-like family member B 10511446 aspartate-beta-hydroxylase 10375137 potassium large conductance calcium-activatedchannel, subfamily M, beta member 1 10528154 predicted gene 6455 ///RIKEN cDNA 4933402N22 gene 10514173 ribosomal protein L34 /// predictedgene 10154 /// predicted pseudogene 10086 /// predicted gene 640410586227 DENN/MADD domain containing 4A 10402648 brain protein 44- like10575745 ATM interactor 10346255 ORM1-like 1 (S. cerevisiae) 10400405nuclear factor of kappa light polypeptide gene enhancer in B- cellsinhibitor, alpha 10528527 family with sequence similarity 126, member A10472738 DDB 1 and CUL4 associated factor 17 10368534 nuclear receptorcoactivator 7 10407543 GTP binding protein 4 10376555 COP9 (constitutivephotomorphogenic) homolog, subunit 3 (Arabidopsis thaliana) 10567297inositol 1,4,5- triphosphate receptor interacting protein-like 210589886 RIKEN cDNA 4930520004 gene 10423593 lysosomal- associatedprotein transmembrane 4B 10577954 RAB11 family interacting protein 1(class I) 10604528 muscleblind-like 3 (Drosophila) 10432675 RIKEN cDNAI730030J21 gene 10385747 PHD finger protein 15 10398240 echinodermmicrotubule associated protein like 1 10511803 RIKEN cDNA 2610029I01gene 10466606 annexin A1 10520304 ARP3 actin- related protein 3 homologB (yeast) 10425903 na 10488709 RIKEN cDNA 8430427H17 gene 10376096acyl-CoA synthetase long- chain family member 6 10429491 activityregulated cytoskeletal- associated protein 10439710 pleckstrinhomology-like domain, family B, member 2 10467110 expressed sequenceAI747699 10536898 interferon regulatory factor 5 10505044 fukutin10605370 membrane protein, palmitoylated 10363669 DnaJ (Hsp40) homolog,subfamily C, member 12 10496727 dimethylarginine dimethylaminohydrolase1 10587683 B-cell leukemia/lymphoma 2 related protein A1a /// B-cellleukemia/lymphoma 2 related protein A1d /// B-cell leukemia/lymphoma 2related protein A1b /// B-cell leukemia/lymphoma 2 related protein A1e10458816 toll like receptor adaptor molecule 2 10513008 Kruppel-likefactor 4 (gut) 10550906 plasminogen activator, urokinase receptor10362674 U3A small nuclear RNA 10473190 DnaJ (Hsp40) homolog, subfamilyC, member 10 10477581 ribosomal protein L5 10571774aspartylglucosaminidase 10395356 anterior gradient homolog 3 (Xenopuslaevis) 10392440 solute carrier family 16 (monocarboxylic acidtransporters), member 6 10352815 interferon regulatory factor 6

REFERENCES

-   Afkarian, M., Sedy, J. R., Yang, J., Jacobson, N. G., Cereb, N.,    Yang, S. Y., Murphy. T. L., and Murphy, K. M. (2002). T-bet is a    STAT1-induced regulator of IL-12R expression in naive CD4+ T cells.    Nature immunology 3, 549-557.-   Ansel, K. M., Djuretic, I., Tanasa, B., and Rao. A, (2006).    Regulation of Th2 differentiation and 114 locus accessibility.    Annual review of immunology 24, 607-656.-   Baron. J. L., Madri, J. A., Ruddle, N. H., Hashim, G., and    Janeway, C. A., Jr. (1993). Surface expression of alpha 4 integrin    by CD4 T cells is required for their entry into brain parenchyma. J    Exp Med 177, 57-68.-   Bell, A. L., Magill. M. K., McKane, W. R., Kirk, F., and    Irvine, A. E. (1995). Measurement of colony-stimulating factors in    synovial fluid: potential clinical value. Rheumatol Int 14, 177-182.-   Berner, B., Akca, D., Jung, T., Muller, G. A., and    Reuss-Borst, M. A. (2000). Analysis of Th1 and Th2 cytokines    expressing CD4+ and CD8+ T cells in rheumatoid arthritis by flow    cytometry. J Rheumatol 27, 1128-1135.-   Bettelli, E., Carrier, Y., Gao, W., Korn, T., Strom, T. B., Oukka,    M., Weiner, H. L., and Kuchroo, V. K. (2006). Reciprocal    developmental pathways for the generation of pathogenic effector    TH17 and regulatory T cells. Nature 441, 235-238.-   Brustic, A., Heink, S., Huber, M., Rosenplanter, C., Stadelmann, C.,    Yu. P., Arpaia, E., Mak, T. W., Kamradt, T., and Lohoff, M. (2007).    The development of inflammatory T(H)-17 cells requires    interferon-regulatory factor 4. Nature immunology 8, 958-966.-   Burchill, M. A., Yang, J., Vogtenhuber, C., Blazar, B. R., and    Farmr, M. A. (2007). IL-2 receptor beta-dependent STAT5 activation    is required for the development of Foxp3+ regulatory T cells. J    Immunol 178, 280-290.-   Campbell. I. K., Rich, M. J., Bischof, R. J., Dunn. A. R., Grail.    D., and Hamilton, J. A. (1998). Protection from collagen-induced    arthritis in granulocyte-macrophage colony-stimulating    factor-deficient mice. J Immunol 161, 3639-3644.-   Campbell, I. K., van Nieuwenhuijze, A., Segura, E., O'Donnell, K.,    Coghill, E., Hommel, M., Gerondakis, S., Villadangos. J. A., and    Wicks, I. P. (2011). Differentiation of inflammatory dendritic cells    is mediated by NF-kappaB1-dependent GM-CSF production in CD4 T    cells. J Immunol 186, 5468-5477.-   Choy, E H., and Panayi, G. S. (2001). Cytokine pathways and joint    inflammation in rheumatoid arthritis. N Engl J Med 344, 907-916.-   Codarri, L., Gyulveszi, G., Tosevski, V., Hesske, L., Fontana. A.,    Magnenat, L., Suter, T., and Becher. B. (2011). RORgammat drives    production of the cytokine GM-CSF in helper T cells, which is    essential for the effector phase of autoimmune neuroinflammation.    Nat Immunol 12, 560-567.-   Cook, A. D., Braine, E. L., Campbell, I. K., Rich, M. J., and    Hamilton, J. A. (2001). Blockade of collagen-induced arthritis    post-onset by antibody to granulocyte-macrophage colony-stimulating    factor (GM-CSF): requirement for GM-CSF in the effector phase of    disease. Arthritis Res 3, 293-298.-   Cope, A. P., Schulze-Koops, H., and Aringer, M. (2007). The central    role of T cells in rheumatoid arthritis. Clin Exp Rheumatol 25,    S4-11.-   Cornish, A. L., Campbell, I. K., McKenzie, B. S., Chatfield, S., and    Wicks, I. P. (2009). G-CSF and GM-CSF as therapeutic targets in    rheumatoid arthritis. Nat Rev Rheumatol 5, 554-559.-   Croxford, A. L., Mair, F., and Becher, B. (2012). IL-23: one    cytokine in control of autoimmunity. European journal of immunology    42, 2263-2273.-   Cua, D. J., Sherlock, J., Chen, Y., Murphy, C. A., Joyce, B.,    Seymour, B., Lucian, L., To, W., Kwan. S., Churakova, T., et al.    (2003). Interleukin-23 rather than interleukin-12 is the critical    cytokine for autoimmune inflammation of the brain. Nature 421,    744-748.-   El-Behi, M., Ciric, B., Dai, H., Yan, Y., Cullimore, M., Safavi, F.,    Zhang, G. X., Dittel, B. N., and Rostami, A. (2011). The    encephalitogenicity of T(H)17 cells is dependent on IL-1- and    IL-23-induced production of the cytokine GM-CSF. Nat Immunol 12,    568-575.-   Gran. B., Zhang, G. X., Yu, S., Li. J., Chen, X. H., Ventura, E. S.,    Kamoun, M., and Rostami, A. (2002). IL-12p35-deficient mice are    susceptible to experimental autoimmune encephalomyelitis: evidence    for redundancy in the IL-12 system in the induction of central    nervous system autoimmune demyelination. J Immunol 69, 7104-7110.-   Gregory, S. G., Schmidt, S., Seth. P., Oksenberg, J. R., Hart, J.,    Prokop, A., Caillier, S. J., Ban, M., Goris, A., Barcellos, L. F.,    et al. (2007). Interleukin 7 receptor alpha chain (IL7R) shows    allelic and functional association with multiple sclerosis. Nature    genetics 39, 1083-1091.-   Greter, M., Helft, J., Chow, A., Hashimoto, D., Mortha, A.,    Agudo-Cantcro, J., Bogunovic, M., Gautier, E. L., Miller, J.,    Leboeuf, M., et al., (2012). GM-CSF controls nonlymphoid tissue    dendritic cell homeostasis but is dispensable for the    differentiation of inflammatory dendritic cells. Immunity 36,    1031-1046.-   Guedez, Y. B., Whittington, K. B., Clayton, J. L., Joosten, L. A.,    van de Loo, F. A., van den Berg, W. B., and Rosloniec, E. F. (2001).    Genetic ablation of interferon-gamma upregulates interleukin-1 beta    expression and enables the elicitation of collagen-induced arthritis    in a nonsusceptible mouse strain. Arthritis Rheum 44, 2413-2424.-   Gutcher, I., and Becher, B. (2007). APC-derived cytokines and T cell    polarization in autoimmune inflammation. J Clin Invest 117,    1119-1127.-   Haak, S., Croxford, A. L., Kreymborg, K., Heppner, F. L., Pouly, S.,    Becher, B., and Waisman, A. (2009). IL-17A and IL-17F do not    contribute vitally to autoimmune neuroinflammation in mice. J Clin    Invest 119, 61-69.-   Hamilton, J. A. (2008). Colony-stimulating factors in inflammation    and autoimmunity. Nat Rev Immunol 8, 533-544.-   Harrington, L. E., Hatton, R. D., Mangan, P. R., Turner, H.,    Murphy, T. L., Murphy,-   K. M., and Weaver, C. T. (2005). Interleukin 17-producing CD4+    effector T cells develop via a lineage distinct from the T helper    type 1 and 2 lineages. Nat Immunol 6, 1123-1132.-   Hofstetter, 1-1.1-1., Ibrahim, S. M., Koczan, D., Kruse, N.,    Weishaupt, A., Toyka, K. V., and Gold, R. (2005). Therapeutic    efficacy of IL-17 neutralization in murine experimental autoimmune    encephalomyelitis. Cell Immunol 237, 123-130.-   Irmler, I. M., Gajda, M., and Brauer. R. (2007). Exacerbation of    antigen-induced arthritis in IFN-gamma-deficient mice as a result of    unrestricted IL-17 response. J Immunol 179, 6228-6236.-   Ivanov, I I. McKenzie, B. S., Zhou. L., Tadokoro, C. E., Lepelley,    A., Lafaille, J. J., Cua, D. J., and Littman, D. R. (2006). The    orphan nuclear receptor RORgammat directs the differentiation    program of proinflammatory IL-17+T helper cells. Cell 126,    1121-1133.-   Kaplan, M. N., Schindler, U., Smiley, S. T., and Grusby, M. J.    (1996a). Stat6 is required for mediating responses to IL-4 and for    development of Th2 cells. Immunity 4, 313-319.-   Kaplan, M. H., Sun, Y. L., Hoey, T., and Grusby, M. J. (1996b).    Impaired IL-12 responses and enhanced development of Th2 cells in    Stat4-deficient mice. Nature 382, 174-177.-   Kolaczkowska, E., and Kubes, P. (2013). Neutrophil recruitment and    function in health and inflammation. Nat Rev Immunol 13, 159-175.-   Komatsu, N., and Takayanagi, H. (2012). Autoimmune arthritis: the    interface between the immune system and joints. Adv Immunol 115,    45-71.-   Korn. T., Bettelli, E., Gao, W., Awasihi, A., Jager. A., Strom, T.    B., Oukka, M., and Kuchroo, V. K. (2007). IL-21 initiates an    alternative pathway to induce proinflammatory T(H)17 cells. Nature    448, 484-487.-   Langrish, C. L., Chen, Y., Blumenschein, W. M., Mattson, J., Basham,    B., Sedgwick, J. D., McClanahan, T., Kastelein, R. A., and    Cua, D. J. (2005). IL-23 drives a pathogenic T cell population that    induces autoimmune inflammation. J Exp Med 201, 233-240.-   Laurence, A., Tato. C. M., Davidson, T. S., Kanno, Y., Chen. Z.,    Yao, Z., Blank, R. B., Meylan, F., Siegel. R., Hennighausen, L., et    al. (2007). Interleukin-2 signaling via STAT5 constrains T helper 17    cell generation. Immunity 26, 371-381.-   Lawlor, K. E., Wong, P. K., Campbell, P. K., van Rooijen, N., and    Wicks, I. P. (2005). Acute CD4+T lymphocyte-dependent    interleukin-1-driven arthritis selectively requires interleukin-2    and interleukin-4, joint macrophages, granulocyte-macrophage    colony-stimulating factor, interleukin-6, and leukemia inhibitory    factor. Arthritis Rheum 52, 3749-3754.-   Lee, L. F., Logronio, K., Tu, G. H., Zhai, W., Ni, I., Mei, L.,    Dilley, J., Yu, J., Rajpal, A., Brown, C., et al. (2012). Anti-IL-7    receptor-alpha reverses established type 1 diabetes in nonobese    diabetic mice by modulating effector T-cell function. Proc Natl Acad    Sci USA 109, 12674-12679.-   Leonard, J. P., Waldburger, K. E., and Goldman, S. J. (1995).    Prevention of experimental autoimmune encephalomyelitis by    antibodies against interleukin 12. J Exp Med 181.381-386.-   Lighvani, A. A., Frucht, D. M., Jankovic, D., Yamanc, H., Aliberti,    J., Hissong, B. D., Nguyen, B. V., Gadina, M., Sher. A., Paul, W.    E., and O'Shea. J. J. (2001). T-bet is rapidly induced by    interferon-gamma in lymphoid and myeloid cells. Proc Natl Acad Sci    USA 98, 15137-15142.-   Liu, X., Lee, Y. S., Yu, C. R., and Egwuagu, C. E. (2008). Loss of    STAT5 in CD4+ T cells prevents development of experimental    autoimmune diseases. J Immunol 180, 6070-6076.-   Lock, C., Hermans, G., Pedotti, R., Brendolan, A., Schadt, E.,    Garren, H., Langer-Gould, A., Stroher, S., Cannella, B., Allard, J.,    et al. (2002). Gene-microarray analysis of multiple sclerosis    lesions yields new targets validated in autoimmune    encephalomyelitis. Nat Med 8, 500-508.-   Lundmark, F., Duvefelt. K., Iacobaeus, E., Kockum, I., Wallstrom,    E., Khademi, M., Oturai, A., Ryder, L. P., Saatchi, J., Harbo, H.    F., et al. (2007). Variation in interleukin 7 receptor alpha chain    (IL7R) influences risk of multiple sclerosis: Nature genetics 39,    1108-1113.-   Manoury-Schwartz, B., Chiocchia, G., Bcssis, N., Ahchsira-Amar, O.,    Batteux, F., Muller, S., Huang, S., Boissier, M. C., and    Fournier, C. (1997). High susceptibility to collagen-induced    arthritis in mice lacking IFN-gamma receptors. J Immunol 158,    5501-5506.-   MeGeachy, M. J., Chen, Y., Tato, C. M., Laurence, A., Joyce-Shaikh,    B., Blumenschein, W. M., McClanahan, T. K., O'Shea, J. J., and    Cua, D. J. (2009). The interleukin 23 receptor is essential for the    terminal differentiation of interleukin 17-producing effector T    helper cells in vivo. Nat Immunol 10, 314-324.-   McInnes, I. B., and Schen, G. (2007). Cytokines in the pathogenesis    of rheumatoid arthritis. Nat Rev Immunol 7, 429-442.-   McInnes, I. B., and Schen, G. (2011). The pathogenesis of rheumatoid    arthritis. N Engl J Med 365, 2205-2219.-   Muller-Ladner, U., Ospelt, C., Gay, S., Distler, O., and Pap, T.    (2007). Cells of the synovium in rheumatoid arthritis. Synovial    fibroblasts. Arthritis Res Ther 9, 223.-   Muller, J., Sperl, B., Reindl, W., Kiessling, A., and Berg, T.    (2008). Discovery of chromone-based inhibitors of the transcription    factor STAT5. Chembiochem: a European journal of chemical biology 9,    723-727.-   Nurieva, R., Yang, X. O., Martinez, G., Zhang, Y., Panopoulos, A.    D., Ma, L., Schluns, K., Tian, Q., Watowich, Jetten. A. M., and    Dong, C. (2007). Essential autocrine regulation by IL-21 in the    generation of inflammatory T cells. Nature 448, 480-483.-   Okada. Y., Wu, D., Trynka, G., Raj, T., Terao, C., Ikari, K., Kochi,    Y., Ohmura, K., Suzuki, A., Yoshida, S., et al. (2014). Genetics of    rheumatoid arthritis contributes to biology and drug discovery.    Nature 506, 376-381.-   Pernis, A. B. (2009). Th17 cells in rheumatoid arthritis and    systemic lupus erythematosus. J Intern Med 265, 644-652.-   Plater-Zyberk. C., Joosten, L. A., Helsen, M. M., Hepp, J.,    Baeuerle, P. A., and van den Berg, W. B. (2007). GM-CSF    neutralisation suppresses inflammation and protects cartilage in    acute streptococcal cell wall arthritis of mice. Ann Rheum Dis 66,    452-457.-   Ponomarev, E. D., Shriver, L. P., Maresz, K., Pedras-Vasconcelos,    J., Verthelyi, D., and Dittel, B. N. (2007). GM-CSF production by    autoreactive T cells is required for the activation of microglial    cells and the onset of experimental autoimmune encephalomyelitis. J    Immunol 178, 39-48.-   Reboldi, A., Coisne, C., Baumjohann, D., Benvenuto, F., Bottinelli,    D., Lira, S., Uccclli, A., Lanzavecchia, A., Engelhardt. B., and    Sallusto, F. (2009). C-C chemokine receptor 6-regulated entry of    TH-17 cells into the CNS through the choroid plexus is required for    the initiation of EAE. Nat Immunol 10, 514-523.-   Segal, B. M., and Shevach, E. M. (1996). IL-12 unmasks latent    autoimmune disease in resistant mice. J Exp Med 184, 771-775.-   Shimoda, K., van Deursen, J., Sangster, M. Y., Sarawar, S. R.,    Carson, R. T., Tripp, R. A., Chu, C., Quelle, F. W., Nosaka, T.,    Vignali, D. A., et al. (1996). Lack of IL-4-induced Th2 response and    IgE class switching in mice with disrupted Stat6 gene. Nature 380,    630-633.-   Sonderegger, I., Iezzi, G., Maier, R., Schmitz, N., Kurrer, M., and    Kopf, M. (2008). GM-CSF mediates autoimmunity by enhancing    IL-6-dependent Th17 cell development and survival. J Exp Med 205,    2281-2294.-   Steinman, L. (2007). A brief history of T(H)17, the first major    revision in the T(H)1/T(H)2 hypothesis of T cell-mediated tissue    damage. Nat Med 13, 139-145.-   Stritesky, G. L., Ych, N., and Kaplan. M. H. (2008). IL-23 promotes    maintenance but not commitment to the Th17 lineage. J Immunol 181,    5948-5955.-   Szabo, S. J., Sullivan, B. M., Stemmann, C., Satoskar, A. R.,    Sleckman, B. P., and Glimcher, L. H. (2002). Distinct effects of    T-bet in TH1 lineage commitment and IFN-gamma production in CD4 and    CD8 T cells. Science 295, 338-342.-   Takeda, K., Tanaka, T., Shi, W., Matsumoto, M., Minami, M.,    Kashiwamura, S., Nakanishi, K., Yoshida. N., Kishimoto, T., and    Akira. S. (1996). Essential role of Stat6 in IL-4 signalling. Nature    380, 627-630.-   Thierfelder. W. E., van Deursen. J. M., Yamamoto, K., Tripp, R. A.,    Sarawar, S. R., Carson, R. T., Sangster, M. Y., Vignali, D. A.,    Doherty, P. C., Grosveld, G. C., and Elle, J. N. (1996). Requirement    for Stat4 in interleukin-12-mediated responses of natural killer and    T cells. Nature 382, 171-174.-   Veldhoen, M., Hirota, K., Westendorf, A. M., Buer, J., Dumoutier,    L., Renauld, J. C., and Stockinger. B. (2008). The aryl hydrocarbon    receptor links TH17-cell-mediated autoimmunity to environmental    toxins. Nature 453, 106-109.-   Veldhoen, M., Hocking, R. J., Atkins, C. J., Locksley, R. M., and    Stockinger, B. (2006). TGFbeta in the context of an inflammatory    cytokine milieu supports de novo differentiation of IL-17-producing    T cells. Immunity 24, 179-189.-   Vermeire, K., Heremans, H., Vandeputte, M., Huang, S., Billiau, A.,    and Matthys, P. (1997). Accelerated collagen-induced arthritis in    IFN-gamma receptor-deficient mice. J Immunol 158, 5507-5513.-   Willenborg, D. O., Fordham, S., Bernard, C. C., Cowden, W. B., and    Ramshaw, I. A. (1996). IFN-gamma plays a critical down-regulatory    role in the induction and effector phase of myelin oligodendrocyte    glycoprotein-induced autoimmune encephalomyelitis. J Immunol 157,    3223-3227.-   Wright, H. L., Moots, R. J., and Edwards, S. W. (2014). The    multifactoral role of neutrophils in rheumatoid arthritis. Nat Rev    Rheumatol 10, 593-601.-   Yamada, H., Nakashima, Y., Okazaki, K., Mawatari, T., Fukushi, J.    I., Kaibara, N., Hori, A., Iwamoto, Y., and Yoshikai, Y. (2008). Th1    but not Th17 cells predominate in the joints of patients with    rheumatoid arthritis. Ann Rheum Dis 67, 1299-1304.-   Yang, X. O., Panopoulos, A. D., Nurieva, R., Chang, S. H., Wang, D.,    Watowich, S. S., and Dong, C. (2007a). STAT3 regulates    cytokine-mediated generation of inflammatory helper T cells. The    Journal of biological chemistry 282, 9358-9363.-   Yang, X. O., Pappu, B. P., Nurieva, R., Akimzhanov, A., Kang. H. S.,    Chung, Y., Ma, L., Shah, B., Panopoulos, A. D., Schluns, K. S., et    al. (2008). T helper 17 lineage differentiation is programmed by    orphan nuclear receptors ROR alpha and ROR gamma. Immunity 28,    29-39.-   Yang, X. P., Ghoreschi, K., Steward-Tharp, S. M., Rodriguez-Canales,    J., Zhu, J., Grainger, J. R., Hirahara, K., Sun, H. W., Wei, L.,    Vahedi. G., et al. (2011). Opposing regulation of the locus encoding    IL-17 through direct, reciprocal actions of STAT3 and STAT5. Nat    Immunol 12, 247-254.-   Yang, Y., Ochando, J. C., Bromberg, J. S., and Ding, Y. (2007b).    Identification of a distant T-bet enhancer responsive to IL-12/Stat4    and IFNgamma/Stat1 signals. Blood 110, 2494-2500.-   Yang, Y. H., and Hamilton. J. A. (2001). Dependence of    interleukin-1-induced arthritis on granulocyte-macrophage    colony-stimulating factor. Arthritis Rheum 44, 111-119.-   Yao, Z., Cui, Y., Watford, W. T., Bream, J. H., Yamaoka, K.,    Hissong, B. D., Li, D., Durum, S. K., Jiang, Q., Bhandoola, A., et    al. (2006). Stat5a/h are essential for normal lymphoid development    and differentiation. Proc Natl Acad Sci USA 103, 1000-1005.-   Zhang, G. X., Gran, B., Yu, S., Li. J., Siglienti, L., Chen, X.,    Kamoun, M., and Rostami, A. (2003). Induction of experimental    autoimmune encephalomyelitis in IL-12 receptor-beta 2-deficient    mice: IL-12 responsiveness is not required in the pathogenesis of    inflammatory demyelination in the central nervous system. J Immunol    170, 2153-2160.-   Zhou, L., Ivanov, I I, Spolski, R., Min, R., Shenderov, K., Egawa,    T., Levy, D. E., Leonard. W. J., and Littman. D. R. (2007). IL-6    programs T(H)-17 cell differentiation by promoting sequential    engagement of the IL-21 and IL-23 pathways. Nature immunology 8,    967-974.-   Zhu, J., and Paul, W. E. (2010). Peripheral CD4+ T-cell    differentiation regulated by networks of cytokines and transcription    factors. Immunological reviews 238, 247-262.

While this invention has been particularly shown and described withreferences to example embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1.-33. (canceled)
 34. A method of treating an inflammatory diseaseassociated with a TNF-α-independent inflammatory pathway, comprisingadministering to a subject in need thereof an effective amount of asignal transducer and activator of transcription 5 (STAT5) inhibitor,wherein the inflammatory disease is mediated by granulocyte macrophagecolony-stimulating factor (GM-CSF)-secreting T-helper (Th-GM) cells,wherein the STAT5 inhibitor reduces serum level of GM-CSF in the subjectand thereby providing a therapeutic benefit to the subject in aTNF-α-independent manner.
 35. The method of claim 34, wherein theinflammatory disease is rheumatoid arthritis.
 36. The method of claim34, wherein the inflammatory disease is multiple sclerosis.
 37. Themethod of claim 34, wherein the T_(H)-GM cells are differentiated fromprecursor CD4⁺ cells in the presence of activated STAT5 and IL-7. 38.The method of claim 34, wherein the STAT5 inhibitor is pimozide.
 39. Themethod of claim 34, wherein the STAT5 inhibitor is CAS 285986-31-4. 40.The method of claim 34, wherein the STAT5 inhibitor is an antibody thatspecifically binds to STAT5.
 41. The method of claim 34, wherein theSTAT5 inhibitor is an antisense nucleic acid, small interfering RNA,short hairpin RNA, or microRNA.
 42. A method of treating rheumatoidarthritis, comprising administering to a subject in need thereof aneffective amount of (A) a STAT5 inhibitor and (B) a therapeutic agentthat inhibits the TNF-α inflammatory pathway.
 43. The method of claim42, wherein the STAT5 inhibitor is pimozide.
 44. The method of claim 42,wherein the STAT5 inhibitor is CAS 285986-31-4.
 45. The method of claim42, wherein the STAT5 inhibitor is an antibody that specifically bindsto STAT5.
 46. The method of claim 42, wherein the STAT5 inhibitor is anantisense nucleic acid, small interfering RNA, short hairpin RNA, ormicroRNA.
 47. The method of claim 42, wherein the therapeutic agent thatinhibits the TNF-α inflammatory pathway is an etanercept, adalimumab,infliximab, golimumab, or certolizumab pegol.
 48. The method of claim42, wherein the therapeutic agent that inhibits the TNF-α inflammatorypathway is prednisone, methotrexate, or tofacitinib.
 49. The method ofclaim 42, Wherein the therapeutic agent that inhibits the TNF-αinflammatory pathway is anakinra, abatacept, rituximab, or tocilizumab.50. The method of claim 42, wherein (A) and (B) are administeredsequentially.
 51. The method of claim 42, wherein (A) and (B) areadministered simultaneously.
 52. The method of claim 42, wherein (A) and(B) are administered in a single dose.